Frequently Asked Question (FAQ) list

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Aug 16 12:20:04 EST 1995


         *************************************************************
         *           Frequently Asked Question (FAQ) list            *
         *            for bionet.molbio.methds-reagnts               *
         *       version 01.16.08.1995 (version.day.month.year)      *
         *************************************************************

                         *** What's New? ***

                                 (1)

I've updated the question on alternative dyes for staining DNA to include
information about the performance of SYBR Green I using the Ames test for
chemical mutagenicity.

                                 (2)

NEW QUESTION ADDED:

Does anyone know of an ftp site or WWW site that has Material Safety Data 
Sheet (MSDS) information?

To get to an alphabetical listing of Material Safety Data Sheets, you can use
gopher by typing the following:
 
gopher atlas.chem.utah.edu
 
--> 12. Material Safety Data Sheets/
 
Or, if you have access to Mosiac or Netscape, you can try any of the following
WWW locations:
 
http://physchem.ox.ac.uk/MSDS/
http://www.fisher1.com/Fisher/Alphabetical_Index.html
http://www.pp.orst.edu
http://unixg.ubc.ca:780/~bchydro/bchydro.html
gopher://atlas.chem.utah.edu/11/msds"
gopher://ginfo.cs.fit.edu:70/1m/safety/msds
gopher://atlas.chem.utah.edu/11/MSDS

                                 (3)

NEW QUESTION ADDED:

Should we break up the methods-reagnts group into subsets with one being
exclusively on the polymerase chain reaction (PCR)?

Some people feel the need to break up the methods newsgroup because the traffic
has increased dramatically over the past year. However, this is mostly a problem
for people who get the posted articles by e-mail and not through a threaded
newsreader. The first suggestion if you are having overload problems is to find
your system administrator and have him/her set you up correctly with newsreader
software.

There are definite advantages to not breaking up bionet.molbio.methds-reagnts.

1.) Most methods and techniques discussed are ubiquitous to all molecular biology.
People with diverse backgrounds are all meeting in one place and this gives a
great spread of interests and approaches to solving problems. If broken up, we
lose major contributors and therefore their insights.

2.) With many different groups that discuss methods, people begin to cross-post
messages in order to reach a wider audience. Cross-posting then becomes so
rampant that you might as well be subscribed to only one newsgroup anyway. If
you are subscribed to a few different groups, you end up re-reading most of the
postings. This becomes tediously boring and wastes a huge amount of time and
effort.

                                 (4)

Many of the questions asked frequently on methds-reagnts can be found answered
within the package of TIBS articles which is available from me. To date, I've
mailed out over 800 packages which were requested.  Most published articles
more than six months old are archived at my ftp site at ftp.ncifcrf.gov in
ASCII format and can be retrieved by anonymous ftp as
pub/methods/TIBS/MONYR.txt, where MON is the month and YR is the year of
publication.  They can also be read directly by using a WWW browser/surfer.
To do this, go to my homepage listed on my .sig file and click on the `README
for the TIBS archive', then click on the BiBTeX identifier key on the list of
articles in order to view the document.

*******************************************************************************
A list of links to molecular biology resources has been added to my homepage
                  http://www-lmmb.ncifcrf.gov/~pnh/
*******************************************************************************

                    *** IMPORTANT!! READ THIS ***

Please DO NOT post a message to the newsgroup asking to be subscribed or
unsubscribed, or send a test message which allows you to see a posting you made
without saying anything. This is a tremendous waste of time for readers and
can be extremely annoying. A test message should be posted to misc.test where
receipt of your message by various machines around the world will be
acknowledged through e-mail. Thousands of people do this every day!
There is now a file named pub/methods/how.to.subscribe at my ftp site listed
below which explains how to subscribe and unsubscribe to METHODS-AND-REAGENTS
by e-mail.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - *
* - - -  Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - *
*******************************************************************************

                     Frequently Asked Questions:

1.  Where can I find the FAQ list for bionet.molbio.methds-reagnts?
2.  Where are the reviews for current topics located?
3.  Are the past articles published in TIBS available on the net?
4.  How do I search for past articles posted to bionet.molbio.methds-reagnts?
5.  What are some good reference books for methods in molecular biology?
6.  Where can I get information about the genotypes and phenotypes of common
    E. coli strains and yeast strains used in molecular genetics?
7.  How can I access MEDLINE?
8.  How do I amplify pBR322 plasmid using chloramphenicol?
9.  If a plasmid has more than one site for a particular restriction
    enzyme, is there some way to get the enzyme to cut at only one site?
10. Why is some plasmid DNA only partially cut by restriction enzymes such as
    BclI, ClaI, or XbaI?
11. How I do prepare powdered silica for DNA purification, with the associated
    solutions?
12. How do I use powdered silica to isolate DNA from agarose gels?
13. How do I use silica powder to prepare plasmid DNA for sequencing?
14. Is there a simple subcloning method for plasmid construction?
15. How do I transform or electroporate E. coli cells with plasmid constructs?
16. Is it possible to clean and re-use electroporation cuvettes?
17. What is PCR?
18. What are some good references for PCR?
19. How should I select a set of primers to use for PCR?
20. What kinds of programs are available for designing PCR primers?
21. What is "Hot-start" PCR?
22. What is AP-PCR or RAPD PCR?
23. What is "Touchdown" PCR?
24. Is there a simple method to sequence lambda, M13, or plasmid clones
    using PCR? 
25. What is solid-phase sequencing?
26. What is cycle sequencing?
27. What is the easiest and most cost efficient means to remove the Dye
    Deoxy-terminators for automated sequencing after cycle sequencing?
28. Is there a sureshot method for double-stranded plasmid sequencing?
29. How do I get my sequencing gel from sticking to the glass plate?
30. Do I need to fix my sequencing gel prior to drying onto paper?
31. Are there alternatives to ethidium bromide for staining nucleic acids within
    agarose gels?
32. How do I calculate an extinction coefficient for a protein with known
    sequence for quantification by uv-measurements?
33. Does anyone know of an ftp site or WWW site that has Material Safety Data 
    Sheet (MSDS) information?
34. Should we break up the methods-reagnts group into subsets with one being
    exclusively on the polymerase chain reaction (PCR)?
35. Is there a list of e-mail addresses for technical service representatives?


                Answers to Frequently Asked Questions:

================================================================================

1.  Where can I find the FAQ list for bionet.molbio.methds-reagnts?

This file can be retrieved by FTP or read through a WWW hypertext link such
as Mosaic. It is available by anonymous FTP from ftp.ncifcrf.gov as the file
pub/methods/FAQlist, by anonymous FTP from net.bio.net as the file
pub/BIOSCI/METHDS-REAGNTS/METHODS.FAQ, by Mosaic at:
http://www-lmmb.ncifcrf.gov/~pnh/, or upon e-mail request to
pnh at ncifcrf.gov

My intention for creating this FAQ list is not to attempt a comprehensive
review of the subjects discussed within the newsgroup bionet.molbio.methds-
reagnts, but rather to provide a quick resource for first time users. Many
of the questions answered below are asked by people new to the group on a
recurring basis.

Citations of this file may be made as:

Hengen, Paul N. (1995) "Frequently Asked Question (FAQ) list for
bionet.molbio.methds-reagnts" version number NN.DD.MM.19YY * available via
anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist or upon request
by e-mail to pnh at ncifcrf.gov

* The version number is designated as: version.day.month.year
  [NN=number, DD=day, MM=month, and YY=year].

If anyone would like to make additions, corrections, or suggestions to
improve this FAQ list, please write to me at pnh at ncifcrf.gov

================================================================================

2.  Where are the reviews for current topics located?

To review the more interesting topics discussed in the newsgroup, a joint
project has been undertaken between the Elsevier journal Trends in Biochemical
Sciences (TIBS) and BIOSCI.  Beginning in the November 1993 issue of TIBS, I
have been publishing a unique monthly column called `Methods and reagents',
consisting of highlights of discussions and bits of reviews originally posted
to the newsgroup bionet.molbio.methds-reagnts.  Each column focuses on one or
two topics of general interest.  This effort represents an exciting new step
combining electronic and hardcopy publication.  A photocopied package of all
the published articles is available from the author and can be requested by
e-mail. In addition, the manuscripts of the articles released to the public
can now be downloaded from my FTP site in ASCII format.

When reading these articles, it must be kept in mind that this column is not
only meant to discuss trends and interesting unpublished observations, but is
also used to stimulate further discussion without constantly bringing newcomers
up to speed. The articles are not comprehensive reviews per se, but are
designed to keep down the noise level and remove extraneous traffic from
clogging the newsgroup.

If you wish to discuss past topics, it may be fruitful to read the referenced
material beforehand. As always, new articles will appear and discussion topics
will resurface. If you want to discuss newly found information regarding a past
discussion, please post a correct and complete reference so others can follow.
Nothing is more frustrating than looking for an article, only to find that the
person who posted it gave the wrong volume number.

The introductory article in the November 1993 issue explains in detail how to
become involved in the newsgroup and how to subscribe/unsubscribe by e-mail;
however, the ftp site published there pointing to where the FAQ list is located
has been changed to ftp.ncifcrf.gov - I'm very sorry for any inconvenience this
has caused. In addition, there is now a file at the FTP site which tells you
how to subscribe and unsubscribe to METHODS AND REAGENTS.

================================================================================

3.  Are the past articles published in TIBS available on the net?

I have set up the archive site so that the published TIBS articles are now
available through any WWW browser and can be viewed and/or downloaded in
ASCII format.  My agreement with Elsevier Publishing Company prohibits release
of the manuscripts for several months after the publication date. Therefore,
past TIBS articles will appear intermittantly, depending on the publication
date and when I'm able to get them onto the FTP site. Also, a list of the past
and future articles written in BiBTeX format is available by anonymous FTP from
ftp.ncifcrf.gov as file pub/methods/TIBS/tibs.bib

================================================================================

4.  How do I search for past articles posted to bionet.molbio.methds-reagnts?

Before asking a question, look on the FAQ list for an answer! If your topic of
choice has not been covered in the FAQ list, then it may have already been
reviewed in TIBS. Look for a listing of articles within the file tibs.bib at
the FTP site. If that fails to satisfy your needs, the next thing to do is
search the archives of all posts made to bionet.molbio.methds-reagnts.  FTP
archives of all the BIOSCI/bionet messages are available at net.bio.net
[204.31.212.2] in /pub/BIOSCI which you can look at or e-mail to youself by
searching for keywords. There is a hypertext search by keyword item on my
homepage.  One other very easy way to search the archives is by using a GOPHER
client. If you have gopher, point your GOPHER at net.bio.net and follow the
prompts. To do this type gopher net.bio.net If you don't have GOPHER, you can
download it for free over the net. To get a copy of GOPHER or to learn how to
use it, download the gopher-faq file via anonymous FTP from
rtfm.mit.edu:/pub/usenet/news.answers/gopher-faq

If you don't know how to use FTP, you can also send an e-mail message to
mail-server at rtfm.mit.edu with "send usenet/news.answers/finding-sources"
in the body to find out how to do FTP by e-mail.

The FAQ list for GOPHER containing common questions and answers about the
Internet Gopher, a client/server protocol for making a world wide information
service, is usually posted to comp.infosystems.gopher, comp.answers, and
news.answers every two weeks.

New users of BIOSCI/bionet may want to read the "Frequently Asked Questions" or
"FAQ" sheet for BIOSCI. The FAQ provides details on how to participate in these
forums and is available by anonymous FTP from net.bio.net [204.31.212.2] in
pub/BIOSCI/biosci.FAQ. It may also be requested by sending e-mail to
biosci-server at net.bio.net by sending the command "info faq" in the body of an
e-mail message.  Any text placed on the Subject: line of your message is
ignored, so please be sure to put the "info faq" command in the main body of
your e-mail message.  The FAQ is posted the first of each month to the
BIONEWS/bionet.announce newsgroup along with the BIOSCI information sheet and
the list of changes to the newsgroups during the preceding month.

If you experience any problems locating the archives, please contact
biosci-help at net.bio.net for any help.

================================================================================

5.  What are some good reference books for methods in molecular biology?
 
There are many books which provide details and recipes for molecular cloning
techniques. The following is a list of reference books which are commonly
available...

Miller, J. H. 1992. A short course in bacterial genetics: A laboratory manual
and handbook for Escherichia coli and related bacteria. Cold Spring Harbor
Press, Cold Spring Harbor, New York.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.
Smith, and K. Struhl. 1991. Current Protocols in Molecular Biology.  John Wiley
and Sons, New York.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York.

Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic Methods in Molecular
Biology. Elsevier, New York.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York.

Rodriguez, R. L., and R. C. Tait. 1983. Recombinant DNA techniques: An
introduction. Benjamin/Cummings Publishing Co., Inc. Menlo Park, CA.

Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York.

Glick and Pasternak, 1994. Molecular Biotechnology. Principles and Applications
of Recombinant DNA. Published by the Amer. Soc. Microbiol.

Promega Corporation provides a detailed manual for many procedures called
"Promega Protocols and Applications Guide".

================================================================================

6.  Where can I get information about the genotypes and phenotypes of common
    E. coli strains and yeast strains used in molecular genetics?

This book contains much information about E.coli strains and how to acquire
them:

Miller, J. H. 1992. A short course in bacterial genetics: A laboratory manual
and handbook for Escherichia coli and related bacteria. Cold Spring Harbor
Press, Cold Spring Harbor, New York.

The following provide detailed descriptions of many E. coli strains:

Bachmann, B. J. 1990. Linkage map of Escherichia coli K-12, Edition 8.
Microbiol. Rev. 54:130-197.

"Derivations and Genotypes of Some Mutant Derivatives of _Escherichia coli_
K12".  Chapter 72 (pp 1190-1224) in volume 2 of _Escherichia coli and
Salmonella Typhimurium. Cellular and molecular biology_, edited by F. C.
Neidhardt et al.  Publ by American Society for Microbiology, Washington,
D.C., 1987.
 
You can write to Barbara Bachmann to order strains or just to get
more information. She is very helpful in finding the particular
strains for your needs. You can also get information by e-mail to
mary at fetalpig.biology.yale.edu. Each bacterial strain will come as
a glycerol swab with a complete genotype outline. Contact:

Dr. Barbara J. Bachmann
E. coli Genetic Stock Center
Department of Biology
Yale University
P.O. Box 6666
New Haven, CT 06511-7444
phone (203) 432-3535

You may access the database by pointing your gopher at cgsc.biology.yale.edu.
For more information about the database or to request strains, contact:

Mary Berlyn
Phone: (203) 432-9997 
E-mail: mary at cgsc.biology.yale.edu
Snail mail: E.coli Genetic Stock Center, Dept. of Biology, 
355 OML, PO Box 6666, Yale University, New Haven, CT 06511

The American Type Culture Collection provides many species of bacteria as well
as other types of cell lines. Their catalog is full of helpful information
concerning the products they supply. You can get more information by
contacting:

American Type Culture Collection
12301 Parklawn Drive
Rockville, Maryland 20852 USA
Toll Free Orders: 1-800-638-6597
phone: (301) 881-2600
FAX: (301) 231-5826
Telex: 908768

You can access the ATCC catalog via modem. The number is 800-647-4710 use
vt100 2400b 8,1,N Login:common  Psswd:common

You can telnet culture.atcc.org (login = SEARCH, password = COMMON) or you can
access ATCC by gopher at:  gopher culture.atcc.org If you still have problems,
you can contact gopher at atcc.org or email to help at atcc.org or help at atcc.nih.gov

There are other databases of strains available on the internet, but they may
require a fee. You can get more information by sending e-mail to the Microbial
Strain Data Network (MSDN) at the address MSDN at CGNET.COM

Catalogs from New England Biolabs, Boehringer Mannheim, Promega, and Bethesda
Research Labs (B.R.L.) all have reference sections that are worth looking at.
B.R.L. publishes FOCUS, a quarterly newsletter with much information about
techniques used in molecular biology.  If you phone them at 800-828-6686 they
will provide you with back-issues of their publications.

For information on Yeast, contact the Yeast Genetic Stock Center:

Yeast Genetic Stock Center
University of California
Department of Microbiology
102 Donner Laboratory 3206
Berkeley, CA  94720-3206
Phone:510-642-0815
Fax: 510-642-8589
E-mail: ygsc305 at violet.berkeley.edu (Rebecca Contropoulo - Curator)

================================================================================

7.  How can I access MEDLINE?

First, you'll need to get an account on MEDLARS.

MEDLARS Management Section Service Desk: 1-800-638-8480 and 1-301-496-6139.
NLM service inquiries:  1-301-496-6308 and 1-800-272-4787. 

Accounts for MEDLARS are made through NTIS (Natl. Tech. Info. Serv.) in
Springfield, Virginia.  They list 703-487-4650 as a telephone number and
703-487-4650 as a FAX number for ordering.  In the US the, NTIS lists a
one-stop -shopping number for NLM services of 1-800-423-9255.  Once you fill
out the Online Billing Agreement with the NTIS, you get a userid and password
from MEDLARS.

Unless you're quite expert with MESH terms and MEDLINE searching, you should
order a copy of GRATEFUL MED 6.0. You can then formulate your search offline
and then telnet to medlars.nlm.nih.gov

You can also access Medline by way of Paperchase, a dial-up service available
through a modem. They provide a 24 hr. toll-free number for questions.  In the
U.S. you can call 800-722-2075. From Canada, you can call collect 617-278-3900.
You can also access Paperchase through telnet to biotechnet.com if you open an
account with them first. The number for BioTechNET is 508-655-8282. You can get
more information about this by sending e-mail to biotechnet at biotechnet.com.

Another  convenient on-line bibliographic database is UnCover, available by
telnet to database.carl.org.  It has a klunky interface, but seems to give
adequate coverage of the literature.  To use it, telnet to database.carl.org
and access the "Uncover" service.  Searching is free.  Moreover (also for
free), you can set up a user profile and have journal contents automatically
emailed to you as they become available.  For payment, they offer to fax copies
of any article to you.  You can set up an account and then just indicate which
articles to have sent to you.

For information regarding database availability and international database
centers, contact:

Director for International Programs
National Library of Medicine
8600 Rockville Pike
Bethesda, Maryland 20894

                --- International MEDLARS Centers ---

Australia:
            National Library of Australia
            OZLINE
            Canberra A.C.T. 2600
            Australia

Canada: 
            Canada Institute for Scientific and Technical Information
            Health Sciences Resource Center
            National Research Council
            Ottawa, Canada K1A 0S2

China:   
            Chinese Academy of Medical Sciences
            Institute of Medical Information
            3 Yabao Road, Chaoyang District
            Beijing, China

Egypt:
            Academy of Scientific Research and Technology
            Centre for Educational Technology
            21, Abdel Aziz Al Seoud Street
            El Rhoda
            Cairo, Egypt

France: 
            Institut National de la Sante et de la Recherche Medicale
            Information Medicale Automatisee
            Centre de Documentation de L'INSERM
            78 Rue du General Leclerc
            94270 Le Kremlin-Bicetre, France

Germany:
            German Institute for Medical Documentation and Information
            5000 Koln 41, Postfach 420580
            West Germany

India:
            National Informatics Centre
            A-Block, CGO Complex
            Lodi Road, New Delhi 110003
            India

Italy:
            Ministry of Health
            Istituto Superiore di Sanita
            Vialo Regina Elena 299
            00161 Rome, Italy

Japan:
            The Japan Information Center of Science and Technology
            5-2 Nagatacho - 2 Chome
            Chiyoda-ku Tokyo
            C.P.O. Box 1478
            Tokyo, Japan

Kuwait:
            Ministry of Public Health
            Arab Centre for Medical Literature
            P.O. Box 5225
            Safat, Kuwait

Mexico:
            Ministry of Health and Welfare
            Centro Nacional di Informacion
            y Documentacion en Salud
            Avda. INSURGENTESSUR #1397-2 Piso
            Col. Mixcoac Insurgentes
            CO 93829 Mexico D.F., Mexico

South Africa:

            South African Medical Research Council
            Institute for Biomedical Communication
            P.O. Box 70
            Tygerberg 7505, South Africa

Sweden:
            Karolinska Institute
            Medical Information Center
            Box 60201
            S-204-01 Stockholm, Sweden

Switzerland:

            Swiss Academy of Medical Sciences
            Documentation Center
            CH-3000 Bern 9, Waldheimstrasse 20
            Berne, Switzerland

United Kingdom:

            The British Library
            Bibliographic Services Division
            2 Sheraton Street
            London W1V 4BH, United Kingdom


Some helpful books are:

Albright, R. G. 1988. A basic guide to online information systems for
health care professionals. Information Resource Press. Arlington, VA.

Feinglos, S. J. 1985. MEDLINE: A basic guide to searching. Medical
Library Association, Inc. Chicago, IL.

================================================================================

8.  How do I amplify pBR322 plasmid using chloramphenicol?

pBR322 is probably the most well known cloning vector among molecular
biologists. A complete review of it's construction can be found here:

Bolivar, F. 1988. Plasmid pBR322: The multipurpose cloning vector.
B.R.L. FOCUS 10:61-64.

The pUC series of cloning vectors provide a number of additional
features including oriented multiple cloning sites. See:

Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived
system for insertion mutagenesis and sequencing with synthetic
universal primers. Gene 19:259-268.

This is the procedure to amplify pUC and pBR based vectors for
large scale plasmid preps. The procedure is directly from Maniatis:

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.

Innoculate a colony from your plate into 10ml of LB broth containing
an appropriate antibiotic, probably ampicillin for pUC plasmids.
Other rich media such as 2xYT, as well as minimal media can also be used. 
Incubate with shaking at 37 degrees overnight.

The next morning add 0.1ml of your overnight culture to 25ml of LB
with  antibiotic (ampicillin) in a 100ml flask. Incubate at 37 degrees
with shaking until the culture is at late log. Usually a couple of
hours is enough. Add 25ml of your fresh late log culture to 500ml of LB
with antibiotic (ampicillin) which has been prewarmed to 37 degrees.
Use a 2L flask for good aeration. Incubate with shaking for exactly
2.5 hours at 37 degrees. Add chloramphenicol to the culture to a final
concentration of 150ug-170ug/ml. If you use spectinomycin the final
concentration should be 300ug/ml. 30ug/ml of chloramphenicol can also
be used. This seems to work just as well as the higher concentration.
Incubate with vigorous shaking for 12-16 hours. If your timing is OK
an overnight incubation will do. Don't worry if the culture turbidity
drops during this incubation, but don't leave the culture for much
longer or you may get too much cell lysis and plasmid yield will drop.
Spin your culture and isolate the plasmid by conventional methods.

There is a mutation in pUC plasmids that increases copy number, but you
can still amplify the plasmids. Most cloning vectors (pUC and pBR
included) have a relaxed replication and don't use the short lifetime host
proteins used in host chromosome replication. So by adding antibiotics
such as chloramphenicol or spectinomycin (which inhibit protein
synthesis) you stop chromosomal replication and cell division without
stopping plasmid replication. The end result is greatly increased
plasmid yield.

================================================================================

9.  If a plasmid has more than one site for a particular restriction
    enzyme, is there some way to get the enzyme to cut at only one site?

Here is a way to use ethidium bromide in restriction digestions to make partial
digests. See:

Parker, R. C., R. M. Watson, and J. Vinograd. 1977. Mapping of closed circular
DNAs by cleavage with restriction endonucleases and calibration by agarose gel
electrophoresis. Proc. Natl. Acad.  Sci. U.S.A. 74:851-855.

These workers used empirical tests to determine the optimal concentrations of
EtBr and enzyme.  A typical reaction is 1ug plasmid +5-20ug EtBr + 2-10 Units
enzyme in 20 ul. These workers also varied the temperature from 4 to 55
degrees, and they found that higher was better.

The technique works well only for *some* enzymes. The paper describes the use
of HindIII and HpaI. Other enzymes are slowed, but not fully inhibited after
the first cut.

You *never* get exclusively full length linears, but EtBr does help getting
linears over just limiting enzyme.

For routine partials without ethidium bromide, make serial dilutions of enzyme
into DNA and run all the products out on an agarose gel with good molecular
weight markers such as the 1 kb ladder from BRL. Usually between 0.5 Units of
enzyme per ug DNA and 100x less gives reasonable partial digestion.

It is MUCH easier to use partial digests if you cut fully with one enzyme, then
do partial digests (by dilution) with the second enzyme.  That way (since you
know the location of the first cut) you can identify the appropriate partial
with ease, AND since the two ends are different you can force clone it into
vector cut with two enzymes.

================================================================================
 
10. Why is some plasmid DNA only partially cut by restriction enzymes such as
    BclI, ClaI, or XbaI?
 
These enzymes are known to be inhibited (blocked) by methylation of adenosine
(DAM) or cytosine (DCM) residues within their recognition site.  The
methylations are caused by site specific methylases (DAM or DCM) in the E.coli
host. For example, strains with DAM or DCM include DH1, HB101, and JM109.  DAM
methylates at the adenine in the sequence GATC and DCM methylates at the second
cytosine within CCWGG. Inhibition is context specific, so not all methylated
recognition sites are blocked, but only those having the methylated residue in
close proximity to the protein binding site. One way to overcome problems with
methylated restriction sites is to (re)transform a dam-/dcm- strain such as
JM110 or DM1 with the respective plasmid and use DNA isolated from this strain
for the particular restriction reactions.  Most, if not all, enzyme suppliers
give appropiate comments in their product data sheets or in their catalogs.
Some suppliers even include charts and/or tables of methylation sensitivity.
Restriction enzymes which may be blocked by overlapping Dam-Methylation can be
found listed in the reference guide of the B.R.L. catalog.(page R-57 of the
1993-1994 catalog).
 
A recent comprehensive table can also be found in:
 
Nelson, M., and M. McClelland. 1991.  Site-specific methylation: effect on DNA
modification methyltransferases and restriction endonucleases.  Nucleic Acids
Res. 19:2045-2071.
 
Nelson, M., and M. McClelland. 1989.  Effect of site-specific methylation on
DNA modification methyltransferases and restriction endonucleases.  Nucleic
Acids Res 17:389-415.
 
================================================================================

11. How I do prepare powdered silica for DNA purification, with the associated
    solutions?

PREPARATION OF GLASS POWDER:

Recipe #1:
Use silica powder: Possible sources include:
    1.  Sigma Chemical Co. (St. Louis, MO).  "Silica"; do not used "fumed 
        silica".
    2.	Cutter Ceramics (11908 Old Baltimore Pike, Beltsville, MD 20705).  
        Ask for 325 mesh powdered flint glass fines.

Resuspend 400 g of silica powder in 800 ml ddH2O in a 2 liter flask.  Stir for
60 mins.  Allow to settle for 90 mins.  Take the SUPERNATANT (which contains
the "fines" of interest) and pellet in Sorvall (GSA rotor, 10 mins at 6000
rpm).  Resuspend pellet in 200-300 ml ddH2O.  Add nitric acid to 50 %.  Bring
close to boiling in fume hood.  Allow to cool.  Pellet silica as before, wash
pellet 4-6 times with ddH2O (check that the pH returns to neutral).  Store
final pellet as 50 % slurry in ddH2O.  Store at -70 deg C, working aliquot at 4
deg C.

Recipe #2:

Do a size (1-g sedimentation) cut, taking the powder that settles out between 2
mins and 60 mins; then boil it all in 10 volumes of 50% nitric acid for an
hour, and wash extensively.


BINDING AND WASH SOLUTIONS:

NaI (BINDING) SOLUTION:

Recipe #1: 90.8 g NaI and 1.5 g Na2SO3 in 100 ml H2O.  Filter through 
        Whatman No. 1 filter paper.  Put a dialysis bag containing  0.5 g 
        Na2SO3 into bottle to keep solution saturated.  Store, 
        foil-wrapped, at 4 C.

Recipe #2:  A saturated solution (at rt) which is approximately a one pound 
        bottle of NaI in 250 ml TE.  Then add a gram of NaSulfite.  This 
        solution dissolves gels quicker and results in a higher binding 
        rate.  Store at RT.  While the solution can be made in water, some 
        batches of NaI need to be buffered.  Buffering the NaI with 20 mM 
        Tris pH 7.5 prevents the lack of DNA binding caused by alkaline NaI 
        solutions.

Recipe #3:  A solution saturated in both NaI and Na2SO3 at room 
        temperature.  Store at room temperature.

NEET WASH:	  100 mM  NaCl
                    1 mM  EDTA
                   50 %   EtOH
                   10 mM  Tris pH 7.5

Store at  -20  C.

================================================================================

12. How do I use powdered silica to isolate DNA from agarose gels?

To purify DNA from agarose gel, weigh the gel slice.  Add 2-3 ml NaI solution
per gram of gel.  Incubate at 37-50 deg C, mixing frequently until agarose is
totally dissolved.  Add 1 microliter of glass slurry per microgram of DNA,
mix.  Incubate on ice 5-10 mins, mixing occasionally.  Spin 5-10 secs in
microfuge, remove and discard supernatant.  Wash glass pellet with 250
microliter NaI (or 10 x volume of silica, whichever is larger).  Spin and wash
pellet 2-3 times with NEET wash (same volume).  Dry pellet well, removing all
residual liquid (air dry or use Kimwipe carefully).  Resuspend pellet in H2O or
TE (> 10 microliter) and elute DNA at 50 C for 5-10 mins.  Spin 1 min in
microfuge and remove eluted DNA in supernatant.

DNA is ready for ligation, restriction, radiolabelling etc.

This proceduure is normally used only on gels run in TAE or Tris phosphate
buffer, but it can be used on borate gels if necessary.  One researcher reports
success by melting the DNA for longer than usual.  Another person says, "Add
100mM NaPO4, pH 6.0 to the NaI solution (we make this up as our stock NaI
solution).  This appears to eliminate problems with TBE gels!."   A final
method:  "About 0.2 g of sorbitol dissolved a 100 mg gel slice in 1 ml of NaI
in about 20 minutes"; this evidently works because the sorbitol ties up the
diol binding sites of the borate.

For more information see:

Vogelstein, B., and D. Gillespie. 1979. Preparative and analytical
purification of DNA from agarose. Proc. Natl. Acad. Sci. U.S.A. 76:615-
619.

================================================================================

13. How do I use silica powder to prepare plasmid DNA for sequencing?

The method from Bill Melchior [wmelchior at ntbtox.nctr.fda.gov] is based on
modifications of the Birnboim and Doly alkaline lysis method.  See:

Carter, M. J. and I. D. Milton. 1993. An inexpensive and simple method
for DNA purifications on silica particles. Nucleic Acids Res. 21:1044

Morelle, G. A plasmid extraction procedure on a miniprep scale. 1989.
B.R.L. Focus. 11:7-8.

Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure
for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-
1523.

MATERIALS:

50 mM glucose/10 mM EDTA/25 mM Tris (pH 8) ("GET"); store in refrigerator.
RNase A 10, mg/ml, heat treated; store frozen.
0.2 N NaOH/1% SDS; store at room temperature.
7.5 M ammonium acetate; store refrigerated..
Silica slurry: 50% solution of powdered silica in H20; store refrigerated 
        or frozen.
NaI/Na2SO3 (Na sulfite); store at room temperature.
NEET wash solution: store in freezer.

(The last three items are described in an earlier section.)

PROCEDURE:

Grow 3 ml overnight cultures of plasmid-containing bacteria.

1.  After chilling overnight cultures on ice for 5 minutes, collect the 
    bacteria in 1.5 ml microcentrifuge tubes by centrifugation.  After 
    decanting, centrifuge the tubes 10 seconds and aspirate the remaining 
    liquid.

2.  Resuspend each pellet in 0.1 ml GET.  

3.  Add 2 microliters RNase A, 10 mg/ml.  Mix.  Allow to sit on ice for 30 
    minutes.

4.  Add 0.2 ml NaOH/SDS.  Mix with three sharp inversions.  Incubate on ice 
    10 min.  The solution will become milky as SDS precipitates.

5.  Add 0.15 ml 7.5 M room teperature ammonium acetate.  Mix with three 
    sharp inversions.  The precipitate will become curd-like.  Continue to 
    incubate on ice for at least 30 min.

6.  Centrifuge 5 minutes.

7.  Aspirate into clean microcentrifuge tubes, trying to avoid most of the 
    precipitate.  Recentrifuge for 3 minutes and again aspirate to clean 
    tubes. 

8.  (Optional.  See Notes.)  Add 2 microliters RNase A, 10 mg/ml, and mix.  
    Incubate at 37 deg C for 30 minutes.

9.  Mix silica slurry with saturated NaI/saturated Na2SO3, using 5 micro-
    liters silica suspension per ml NaI/Na2SO3.  Before the silica settles, 
    add 0.9 ml to each sample and mix gently.  Put samples on ice for at 
    least 1 hr.

10. Centrifuge the samples 5 minutes and decant.  

11. Wash the silica pellet three times with 0.2 ml NEET solution, Vortexing 
    each time to suspend and then centrifuging one minute.  After the last 
    wash is decanted, centrifuge 10 seconds and aspirate the remaining 
    liquid.

12. Add 20 microliters 10 mM Tris-HCl (pH 7.5-8)/1 mM EDTA (TE) to each 
    sample, Vortex to suspend, and incubate at 45-55 degrees C for 3 
    minutes.  Centrifuge one minute; aspirate to clean tubes.

13. Repeat step 12, giving a total of about 40 microliters supernatent for 
    each sample.  Centrifuge three minutes and again aspirate to clean 
    microcentrifuge tubes.

14. Remove one microliter of each sample for agarose electrophoresis.

15. If electrophoresis indicates an adequate yield, the samples can be 
    denatured according to the Sequenase protocol, using twice the 
    recommended volumes:  Add four microliters 2 N NaOH, mix, and incubate 
    at room temperature 5 minutes.  Add 16 microliters 5 M ammonium acetate 
    and 0.2 ml EtOH.  Mix, chill the samples, collect by centrifugation, 
    wash with 0.4 ml 70% EtOH at room temperature, and dry.  Dissolve in 7 
    microliters H2O or TE and proceed with sequencing.

NOTES ON PROCEDURE:

1.  Use two successive centrifugations to collect bacteria from 3 ml in 
    a single tube.

3.  Lysozyme may be needed at this step for some bacterial strains.  It is 
    not needed for E. coli strains AB1157, DH1, or HB101.

4,5.Vigorous mixing at these steps is not only unnecessary, but leads 
    to breakage for bacterial DNA, with subsequent contamination of the 
    plasmid.  Three sharp snaps of the wrist is adequate.  (The mixture 
    will not appear uniform -- do not worry about it.)

7.  It is easier to perform a second centrifugation to remove any 
    precipitate that is carried over than it is to avoid the precipitate 
    completely.

8.  RNA digestion during the first incubation is variable, so a second 
    RNase treatment is sometimes needed.  An alternative would be to treat 
    with RNase just before denaturation if gel electrophoresis shows it to 
    be necessary;  RNase added before the denaturation step does not 
    interfere with Sequenase sequencing.  If RNA is BARELY visible on the 
    agarose gel, it doesn't seem to seriously interfere with the 
    sequencing.

9.  If the NaI solution is added to the plasmid preparation first, followed 
    by the glassmilk, the silica clumps and is difficult to resuspend.  
    Mixing the NaI and glassmilk and then adding the mixture to the 
    preparation avoids this difficulty.

13. As noted for step 7, it is easier to remove solid material carried over 
    during the aspiration than to avoid it.  If the silica contains very 
    fine particles, there will be some carry-over even after the second 
    centrifugation, but small amounts do not interfere with Sequenase 
    sequencing.

15. The samples can be stored in the freezer, either in the native state 
    or after denaturation, and either dry or redissolved.

The preparations appear to be dirtier than those produced by other 
protocols; in addition to covalently closed circular plasmid, there are 
open circles, denatured plasmid, and a small amount of linear plasmid, plus 
chromosomal DNA and, sometimes, a small amount of RNA.  Nonetheless, the 
sequences obtained are strong and unambiguous.  The improved sequencing 
results may be due at least in part to improved yields with this method.  
This method has been used to prepare pBR322 for both Sequenase double-
strand sequencing and the PRISM dye terminator cylce sequencing system of 
ABI; denaturation (step 15) is not needed for cycle sequencing.

================================================================================

14. Is there a simple subcloning method for plasmid construction?

Low-melt agarose method from Jim Graham (jgraham at bronze.ucs.indiana.edu):

Cut your vector and insert fragments and run them out on a low melting agarose
gel (eg. SeaPlaque from FMC BioProducts) in TAE buffer.  Excise the bands of
interest on a LONG WAVE UV box making sure to cut the smallest slice possible.
For very low percentage agarose, use a thin layer of standard agaorse (1%) as a
support by pouring it without a comb, setting the comb higher, and pouring your
low melting agarose gel on top once the support layer has set.  Cut this layer
off as well when you excise your bands.  Place  the gel slices in sterile
eppendorfs and melt them at 70C in a beaker. Do not add any buffer or dilute
the slices. Carry this beaker to the -70 freezer and put the samples in an
isopropanol bath. Wait 5 minutes, and then thaw them.  Spin 5 minutes in the
microfuge. The supenatant is your sample for ligation.  Take 2 ul of vector
supernatant and distribute it to 4 screw top eppendorf tubes. Add 3 ul of BRL
ligase buffer (5X w/ PEG), 8 U of NEB ligase for sticky ends, and either 1, 3,
or 7 ul of insert supernatant.  Bring the volume to 15 ul with sterile water.
Incubate at 16 C overnight by emersing the entire tube beneath the suface of a
water bath in the cold room.  In the morning dilute your 15 ul ligation 5X to
75 ul and add 20 ul per 250 ul of Ca/Rb competent cells.

Glass-milk method from Paul N. Hengen (pnh at ncifcrf.gov):

Slice both fragments from the gel and put them together in a tube.  Bind the
DNA fragments to glass-milk, wash 3x with the NEET buffer.  After the final
spin, dry the glass-milk DNA complex and then add 10 ul of water. Put the tube
at 45-50 C for 5 minutes, then flick the tube a few times. Incubate another 5
minutes and then spin out the glass-milk. Put on ice until you remove the DNA
for ligation. (you lose 3 ul due to the glass-milk).  Use 7 ul mixed DNA plus 2
ul 5x ligase buffer plus 1 ul of T4 DNA ligase on ice _OR_ simply add an equal
volume of 2x ligation buffer and 0.5-1.0 ul of ligase. Place at 12-16 C
overnight. Transform 100 ul of competent cells with the entire amount of DNA
and plate onto selective media.

================================================================================

15. How do I transform or electroporate E. coli cells with plasmid constructs?

Transformation:
***************

There are many methods which will give you a number of transformants when you
only need to change hosts or if you recieve plasmid DNA from someone else. A
simple technique is to centrifuge exponentially growing (~2 x 10^8 cfu/ml)
cells at 3000 rpm and then resuspend them in 1:20 volume of cold (4 C) 75mM
CaCl2. Generally, 50-100 ul of concentrated cells are placed in a
microcentrifuge tube. Keep the cells on ice with DNA added, heat shock at 42 C
for 90-120 seconds, add fresh broth to express for about an hour at 37 C. Plate
the undiluted mixture or a microfuge concentrated portion directly onto dry,
selective media.

Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic
resistance in bacteria: genetic transformation of Escherichia coli by R-factor
DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.

Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride
improves the competence of Escherichia coli cells. Gene 6:23-28.

A very quick technique involving a freezing step is:

Takahashi, R., S. R. Valeika, and K. W. Glass. 1992. A simple method of plasmid
transformation of E. coli by rapid freezing. Biotechniques 13:711-715.

For more efficient transformation procedures, you may need to use one of the
more complicated methods published by D. Hanahan:

Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids.
J. Mol. Biol. 166:557-580.

Hanahan, D. 1985. Techniques for transformation of E. coli, p.109-135.  In: D.
M. Glover (ed.), DNA cloning: A practical approach, Volume I, IRL Press,
Oxford.

Other methods are:

Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and K. Arai.
1987.  High-efficiency clonning of full-length cDNA; construction and screening
of cDNA expresion libraries for mammalian. Methods Enzymol. 154:3-28.

Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of
Escherichia coli with plasmids. Gene 96:23-28.

Nishimura, A., M. Morita, Y. Nishimura, and Y. Sugino. 1990. A rapid and highly
efficient method for preparation of competent Escherichia coli cells. Nucl.
Acids Res. 18:6169.

Liu, H. and A. Rashidbaigi. 1990. Comparison of various competent cell
preparation methods for high efficiency DNA transformation. BioTechniques
8:21-25.

Tang, X., Y. Nakata, H.-O. Li, M. Zhang, H. Gao, A. Fujita, O. Sakatsume, T.
Ohta, and K. Yokoyama. 1994.  The optimization of preparations of competent
cells for transformation of E. coli. Nucl. Acids Res. 22:2857-2858.

Electroporation:
****************

Electroporation of E. coli is well established in the scientific literature,
where electro-transformation efficiencies of 10^10 transformants per ug of
supercoiled plasmid DNA can be expected for several E. coli strains.
Advantages of electroporation over chemical methods include ease and speed.

For more information, see:

Dower, W. J., Miller, J. F., and Ragsdale, C. W. 1988. High efficiency
transformation of E.  coli by high voltage electroporation.
Nucl. Acids Res. 16(13):6127-6145.

Many people have used the following protocol with success:

Procedure for High Efficiency Electro-transformation of E. coli 

A.  Preparation of Cells

    1.  Inoculate 1 liter of L-broth(a) with 1/100 volume of a fresh overnight  
        culture.

    2.  Grow cells at 37 C with vigorous shaking to an A 600 of approximately 
        0.5 - 0.7 which shuld give approx. 1 x 10^8 CFU/ml (the best results
        are obtained with cells that are harvested at early- to mid-log phase;
        the appropriate cell density therefore depends on the strain and growth
        conditions). In general, this will be reached 2.5 to 3 hours after
        diluting an overnight culture 1:50.

    *3. To harvest, centrifuge cells in cold centrifuge bottles in a cold rotor 
        at 4000 x g for 15 min.

    *4. Remove as much of the supernatant (medium) as possible.  It is better   
        to sacrifice the yield by pouring off a few cells than to leave any     
        supernatant behind.

    *5. Gently resuspend the pellets in a total of 1 liter of ice-cold 10%      
        glycerol(b) taking care not to lyse them.  Centrifuge as in step 3.

    *6. Resuspend in 0.5 liter of ice-cold 10% glycerol.
        Centrifuge as in step 3.

    *7. Resuspend in ~250 ml of ice-cold 10% glycerol.
        Centrifuge as in step 3.

    8.  Resuspend to a final volume of 3 to 4 ml in ice-cold 10% glycerol.
        The cell concentration should be about 1 - 3 x 10-to-the-10th cells/ml.

    9.  This suspension may be frozen in aliquots on dry ice, and stored at     
        -70 C.  The cells are good for at least 6 months under these           
        conditions.

    *   Keep the cells as close to 0 C as possible (in an ice/water bath)      
        throughout their preparation.

B.  Electro-transformation and Plating

    1.  Gently thaw the cells at room temperature and then immediately place    
        them on ice.  Remove the sterile electroporation cuvettes from their    
        pouches and place on ice.

    2.  In a cold, 1.5 ml polypropylene tube, mix 40 ul of the cell suspension  
        with 1 to 2 ul of DNA (typically 10 pg/ul). [DNA should be in a low     
        ionic strength buffer such as TE(c).]  Mix well and let sit on ice ~0.5 
        - 1  minute.

    3.  Transfer the mixture of cells and DNA to a cold electroporation         
        cuvette, making sure that the liquid is in contact with both metal      
        faces of the cuvette.

    4.  Pulse once at a field strength of 18 kV/cm and a time constant of 4.5   
        to 5.5 milliseconds.

    5.  Remove the cuvette from the chamber and immediately add 1 ml of SOC(d)  
        medium (at room temp.) to the cuvette and quickly, but gently,          
        resuspend the cells with a pipette.  (This rapid addition of SOC after  
        the pulse is very important in maximizing the recovery of               
        transformants.)

    6.  Transfer the cell suspension to a 17 x 100 mm polypropylene tube and    
        incubate at 37 C for 1 hour.  (Shaking the tubes at 225 RPM during     
        this incubation may improve the recovery of transformants.)

    7.  Check and record the pulse parameters.  The time constant should be     
        close to 5 milliseconds.  The field strength can be calculated as the   
        actual volts delivered (kV) / cuvette gap (cm).

    8.  Plate on selective medium.
___________________
 
    
    (a) L-Broth:  1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl.

    (b) 10% Glycerol:  Prepare fresh weekly with sterilized water.  Do not      
        autoclave or filter-sterilize the glycerol solution.

    (c) DNA containing too much salt will make the sample too conductive and    
        cause arcing at high voltage.  TE:  10 mM Tris-HCl pH 8.0, 1 mM EDTA.

    (d) SOC:  2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM   
        KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose.


Caution should be followed if you plan to re-use cuvettes (see the question
pertaining to re-using electroporation cuvettes in this FAQ list).  There are
obvious sterility and cross-contamination risks; since electroporation is the
most efficient E. coli transformation method available, even a minute amount of
DNA from the previous pulse is likely to become a problem.  Less obvious is the
observation that electro-transformation efficiencies are highest in a uniform
electric field (i.e., between two parallel electrodes).  A high-voltage pulse
of a high concentration of E. coli cells causes the cells in contact with the
aluminum surface of the cuvette to actually "bake" onto the electrode.  This
obstruction of the dead cells has the same electrical effect as pitting the
electrodes with either a physical "cleaning" procedure or a harsh chemical
treatment of acid, or base such as bleach, in that the smooth surfaces of the
electrodes are compromised.  The net result is a nonuniform electric field for
the pulse, and a lower transformation efficiency.

Additional bacterial electro-transformation references are avialable from
Connie Rickey of Bio-Rad Laboratories at crickey at haley.genetics.bio-rad.com

================================================================================

16. Is it possible to clean and re-use electroporation cuvettes?

Opinion varies concerning the life of electroporation cuvettes,
but they can usually be re-used 3 - 8 times before they become unsafe.

Here are two methods which can be used for cleaning the cuvettes:

A. Simple Method:

1. Wash out cuvettes with bleach.
2. Rinse 6x with dH2O
3. Rinse with 95% EtOH. 
4. Fill again with 95% EtOH, replace cap, invert, 
   tip off EtOH and dry upside-down in hood.

B. Paranoid Method:

1. Wash out cuvettes with bleach.
2. Rinse 6x with dH2O.
3. Fill with 0.25 M HCl, stand RT, 15' - 2h.
4. Rinse in dH2O, boil in fresh, clean, distilled water for 10'.
5. Remove from bath, immediately rinse in 95% EtOH
   and dry upside down in hood.  Caps can be rinsed in 70% EtOH.

Note: Do not store cuvettes for any length of time in the presence of moisture.

================================================================================

17. What is PCR?

PCR is an acronym which stands for polymerase chain reaction. The PCR technique
is basically a primer extension reaction for amplifying specific nucleic acids
in vitro. The use of a thermostable polymerase allows the dissociation of newly
formed complimentary DNA and subsequent annealling or hybridization of primers
to the target sequence with minimal loss of enzymatic activity. PCR will allow
a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a
million fold so that one can determine its size, nucleotide sequence, etc. The
particular stretch of DNA to be amplified, called the target sequence, is
identified by a specific pair of DNA primers, oligonucleotides usually about 20
oligonucleotides in lenth.  PCR has revolutionized molecular genetics and
continues to be applied to many fields of biology.

================================================================================

18. What are some good references for PCR?

Mullis, K. 1994. The Polymerase chain reaction. Birkhauser Press, Boston,
Mass.

Griffin, H. G. and A. M. Griffin. 1994. PCR Technology: Current Innovations
CRC Press, Boca Raton, Florida.

Mc Pherson, M. J., P. Quirke and G.R. Taylor. 1992. PCR: A Practical Approach
IRL Press.

Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. 
PCR Protocols: A guide to methods and applications. Academic Press, New York.

Erlich, H. A. 1989. PCR technology: Principles and applications for DNA
amplification. Stockton Press, New York.

Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing.
Eaton Publishing Co., Natick, Mass.  (This book contains selected articles on
sequencing PCR products published in the journal BioTechniques up until March
1992)

Bej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Amplification of
nucleic acids by polymerase chain reaction (PCR) and other methods and
their applications. Critical Reviews in Biochemistry and Molecular
Biology 26:301-334.  (Here is an extensive review article with 236 references
regarding PCR)

Linz, U., U. Delling, and H. Rubsamenwaigmann. 1990. Systematic Studies on
Parameters Influencing the Performance of the Polymerase Chain Reaction.
Journal of Clinical Chemistry and Clinical Biochemistry 28:5-13.

There is also a Journal entitled "PCR Methods and Applications" published
quarterly by Cold Spring Harbor Press. You can subscribe to this journal
by writing to:

Cold Spring Harbor Laboratory Press
10 Skyline Drive
Plainview, New York USA  11803-9729

In the U.S. and Canada, you can call 1-800-843-4388. All other locations
can reach them at 516-349-1930 (phone) or 516-349-1946 (FAX).

================================================================================

19. How should I select a set of primers to use for PCR?

See: Innis, M. A., and D. H. Gelfand. 1990. Optimization of PCRs. In:
PCR Protocols: A guide to methods and applications. Academic Press,
New York.
 
Here are some general pointers:

1. Try to keep the primer 50% G-C give or take 15%. If overly G-C rich add 
a string of As or Ts at 5' end; If overly A-T rich, do the same with Gs and Ts.

2. Try to avoid Gs and Cs at 3' end of the primers. This may increase
the chance of forming primer dimers.

3. Avoid self-annealing regions within each primer.

4. Compute Tm as sum of 4 C for G/C and 2 for A/T, then subtract 5 C 
from this value and that is our annealing temp. Naturally, the annealing 
temp will be that of the primer with the lower value. Differences of 4-6 C 
do not seem to affect yield of PCR. Ideally you would like the Tm for each
primer to match and be within the 70-75 degrees C range.

5. A good practice is to check the target DNA sequence if it is known
for mispriming areas. A quick check scanning the sequence of vector for
approximately 70% and above homolgy regions can help prevent obtaining
multiple contaminating bands in your PCR.

6. Use of a computer program may help eliminate the use of a poorly
designed pair of primers.

================================================================================

20. What kinds of programs are available for designing PCR primers?

There are four programs which deal with pcr primer construction which one
can obtain by anonymous FTP and one which will be mailed to you by the author.
The programs are:

1. Pgen - for DOS only

PrimerGen searches strings of amino acid residues in order to reverse-translate
oligonucletide primers of a desired range of lengths and maximum number of
degeneracies.

PrimerGen only works on IBM-PC(TM), XT, AT, PS/2 and compatibles with
EGA or VGA graphics adaptors.  It will not work on computers with CGA or
Hercules(TM) graphics cards. A hard drive is NOT required and PrimerGen will
fit on a 360K 5.25" floppy disk.

PrimerGen contains a sequence editor where amino acid residues are entered.
The amino acid sequence must be ONE fragment and cannot be longer than 70
residues.  The sequence must be in the ONE LETTER CODE and cannot contain
any UNKNOWNS.  After the desired amino acid sequence has been entered have the
option of saving the sequence to a disk. PrimerGen will also accept and
re-edit previously saved sequence files, and also contains a codon preference
table editor. You can get this program by anonymous FTP at
ftp.bio.indiana.edu. It is found in the molbio/ibmpc directory.

2. Primer - Stanford - Sun Sparcstations only 

The program 'primer' is written by Don Faulkner. It helps to find potential
mispriming sites (primer sequences should be designed before running the
program!). The program gives higher weights to matches at the 3'
end of the primer, linearly decreasing them towards the 5' end (the default
is weight=10 for 3' nucleotide decreasing to 1 at nucleotide # 8 from the 3'
end). The program can be used when amplifying *long* fragments from a known
sequence. The program is written in "C" and runs on Sun workstation (Unix).
You can get the program by contacting James Mullins at Stanford University.
[e-mail: jmullins.stanford.edu  phone: (415) 723-0668]

3. Primer - Whitehead -Unix, Vms (and DOS and Mac if you can compile it)

PRIMER is a computer program for automatically selecting PCR primers
written by Steve Lincoln, Mark Daly, and Eric Lander.

PRIMER will run on just about anything which supports a standard C
language compiler.
- IBM PC and compatibles.  A PC/AT (286) class machine or better with 640K of
- Apple Macintosh computers.  A Mac II series or SE/30 with a math coprocessor
- Most Unix workstations including Sun SPARCStation and DEC DECStation systems
- DEC VAX/VMS computers under VMS Version 5 with VAX C.

PRIMER is available from a number of sources:
- You can get PRIMER from another user, provided you strictly follow the terms
- You can download the most recent version off the Internet via anonymous FTP
- You can send a request to:

          PRIMER c/o The Lander Lab,
          Whitehead Institute/MIT
          9 Cambridge Center, Cambridge, MA 02142.

E-mail to "primer at genome.wi.edu" (Internet), or FAX to 617-258-6505
You can also get PRIMER by anonymous FTP from genome.wi.edu in the
distribution/primer.0.4 directory.

4. Amplify - MAC only

This software is for use in designing, analyzing, and simulating
experiments involving the polymerase chain reaction (PCR).  PCR is a
technique used by molecular biologists to amplify highly selected
segments of DNA.

You can obtain a copy of Amplify from 
sumex-aim.stanford.edu via anonymous FTP. Look for the file:

      /info-mac/app/amplify-10.hqx

The author, Bill Engels, can be reached at:

Phone: (608) 263-2213
lab:   (608) 262-5578
Fax:   (608) 262-2976
E-mail: wrengels at facstaff.wisc.edu


5. OSP - Unix, X-windows, Vms, DOS, Mac - by snail-mail only.

OSP is available for free, but the university lawyers require that you
sign a licensing agreement. The legal document is not for the
paperwork faint-at-heart. It is quite long and daunting. If you're
waiting for the legal stuff before starting your experiment, you
may be better off working out a primer by hand.

Here is the abstract from the paper describing OSP, which appeared in 
PCR Methods and Applications 1, 124-128 (1991), along with information
on how to get the program.

ABSTRACT

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA
sequencing and the polymerase chain reaction (PCR).  The user can specify (or
use default) constraints for primer and amplified product lengths, %(G+C),
(absolute or relative) melting temperatures, and primer 3' nucleotides. To help
minimize non-specific priming and primer secondary structure, OSP screens
candidate primer sequences, using user-specifiable cutoffs, against potential
base pairing with a variety of sequences present in the reaction, including the
primer itself, the other primer (for PCR), the amplified product, and any other
sequences desired (e.g., repetitive element sequences in genomic templates,
vector sequence in cloned templates, or other primer pair sequences in
multiplexed PCR reactions).  Base pairing involving the primer 3' end is
considered separately from base pairing involving internal sequences.  Primers
meeting all constraints are ranked by a ``combined score'', a user-definable
weighted sum of any of the above parameters.

OSP is being routinely and extensively used to select sequencing primers for
the  C. elegans genome sequencing project, and human genomic PCR primer pairs
for the Washington University Genome Center mapping project, with success rates
exceeding 96% and 81% respectively. It is available for research purposes from
the authors, at no cost, in both text output and interactive graphics (X
windows) versions.

AVAILABILITY

C language source code for OSP is available (for research purposes only) at no
cost from the authors,  in either the text output version (tested for VAX/VMS,
PC, MAC, and SUN Sparcstations), or interactive X windows graphics version
(tested for SUN Sparcstations).

To obtain OSP please send your postal address either to Phil Green by email,
(pg at genome.wustl.edu) or (preferably) by FAX to (314) 362-2985 c/o Paula,
the secretary handling OSP requests.  You must provide a signed licensing
agreement (which she will send you) and a stamped addressed mailer with
diskette before the program can be sent to you. Sorry for the formalities.

================================================================================

21. What is "Hot-start" PCR?

"Hot-start" PCR is a method that generally produces cleaner PCR products.
Template DNA and primers are mixed together and held at a temperature above the
threshold of non-specific binding of primer to template. All the PCR reaction
components are added for the extention reaction except one critical reagent
(usually the thermostable polymerase).

Just prior to the cycling, the missing component is added to allow the reaction
to take place at higher temperature. Due to lack of non-specific hybridization
of primers to template, the amplified DNA bands tend to be cleaner; the primers
don't have a chance to anneal non-specifically.

This method is difficult to do because the tubes must be kept on a 100C heat
block as your work surface. There are ways to avoid this however. One way is to
quickly cool the tubes on ice while adding the component mix. You can then heat
the tubes on the pre-warmed thermocycler just before adding the last component.
This may not always be successful due to a thermal ramp that may allow
non-specific interactions between primer and template.

Hot starts are also done by creating a physical barrier between the essential
components, eg. primers and template. This barrier may be created by putting a
half-reaction mixture into the bottom of the tube and melting wax over the mix.
The wax used can be "PCR Gems" from Perkin-Elmer/Cetus or any number of
home-grown waxes (e.g. paraffin or Paraplast). Cooling solidifies the wax, and
the missing components can be placed on top. The mixing of the last component
then occurs at high temperature only when the wax melts and the top half-mix is
added by convection currents within the tubes. The PCR then proceeds as a
normal cycle sequence.

Co-solvents have also been used to eliminate artifacts from PCR reactions. For
high fidelity, the specificity of primer to template is desirable. Co-solvents
such as glycerol, DMSO, and formamide, work to provide highly stringent
reactions by changing the Tm of the primer-template hybridization reaction.

Co-solvents have various effects on the thermostablility of the polymerase
enzyme. Glycerol tends to extend the resistance of Taq enzyme to heat
destruction, while formamide lowers enzyme resistance.

In some cases, it may be necessary to add single-strand DNA binding protein in
order to keep DNA with a high GC content from forming secondary structures.
This may also be a problem in cycle sequencing reactions.

See the following references for more details:

Dutton, C. M., C. Paynton, and S. S. Sommer. 1993. General method for
amplifying regions of very high G + C content. Nucleic Acids Research
21:2953-2954.

Blanchard, M.M., Taillon-Miller, P., Nowotny, P., Nowotny, V. 1993.  PCR buffer
optimization with uniform temperature regimen to facilitate automation. PCR
Methods and Applications 2: 234-240.

Rapley, R., S. Flora, and M. R. Walker. 1992. Direct PCR sequencing of murine
immunoglobulin genes using E. coli single-stranded DNA-binding protein. PCR
Methods and Applications 2:99-101.

Chou, Q. 1992. Minimizing deletion mutagenesis artifact during Taq DNA
polymerase PCR by E.coli SSB. Nucleic Acids Research 20:4371.

Wainwright, L. A., and H. S. Seifert. 1993. Paraffin beads can replace mineral
oil as an evaporation barrier in PCR. BioTechniques 14:34-36.

Horton, R. M., Hoppe, B. L., and B. M. Conti-Tronconi. 1994. AmpliGrease:
``hot start'' PCR using petroleum jelly. BioTechniques 16:42-43.

D'Aquila, R. T., Bechtel, L. J., Videler, J. A., Eron, J. J., Gorczyca, P.,
and J. C. Kaplan. 1991. Maximizing sensitivity and specificity of PCR by
preamplification heating. Nucleic Acids Research 19:3749.

Chou, Q., Russell, M., Birch, D. E., Raymond, J., and W. Bloch. 1992.
Prevention of pre-PCR mis-priming and primer dimerization improves
low-copy-number amplifications. Nucleic Acids Research 20:1717-1723.

================================================================================

22. What is AP-PCR or RAPD PCR?

Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD)
are methods of creating genomic fingerprints from species of which little
is known about target sequence to be amplified.

Strain-specific arrays of DNA fragments (fingerprints) are generated by PCR
amplification using arbitrary oligonucleotides to prime DNA synthesis from
genomic sites which they fortuitously match or almost match.  Generally, two
cycles of PCR are performed under conditions of low stringency with a single
random orimer, followed by PCR at high stringency with specific primers.

DNA amplified is this manner can be used to determine the relatedness of
species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).

For more information see:

Welsh, J. and M. McClelland. 1990. Fingerprinting genomes using PCR with
arbitrary primers. Nucl. Acids Res. 18:7213-7218.

Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and
S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are
useful as genetic markers. Nucl. Acids Res. 18:6531-

================================================================================

23. What is "Touchdown" PCR?

Touchdown PCR involves decreasing the annealling temperature by 1 degree C
every second cycle to a 'touchdown' annealing temp which is then used for 10 or
so cycles.  It was originally intended to bypass more complicated optimization
processes for determining optimal annealing temperatures. The idea is that any
differences in Tm between correct and incorrect annealing gives a 2-fold
difference in product amount per cycle (4-fold per degree C). You therefore
enrich for the correct product over any incorrect products.

Another use for this procedure is in determining DNA sequence for a known
peptide sequence. The strategy here is to use two sets of degenerate primers
that match potential coding sequences at the two ends of a peptide of known
sequence. In practice, this requires that you know a stretch of peptide
sequence of only 13 amino acids, with left and right primers of 18 nt (6 a.a.)
and a space in between of one or more nt. Using these degenerate primers, you
do a touchdown PCR. You will get a huge number of products, but you can select
the desired product based on size since you know the exact interprimer distance
from the peptide sequence. The advantage of this technique is that the
touchdown PCR enriches for products containing correct matches between primers
and template. If you clone and sequence a dozen PCR products, you can determine
the correct coding sequence for the peptide, design an oligo for hybridization,
etc. The technique is especially useful for peptide sequences full of ser, lys,
and arg (six codons each).

For more information see:

Don, R. H., P. T. Cox, B. J. Wainwright, K. Baker, J. S. Mattick. 1991.
Touchdown PCR to circumvent spurious priming during gene amplification.
Nucleic Acids Res. 19:4008-

================================================================================

24. Is there a simple method to sequence lambda, M13, or plasmid clones
    using PCR? 

PCR amplification can be can be performed using a phage plaque or bacterial
colony picked directly from an agar plate. This is particularly useful for
confirmation of mutants after site-directed mutagenesis, sequence tagged
site(s) sequence characterization, identification of mutations following random
mutagenesis, etc.

For more information see:

Wang, H.  and A. J. Cutler. 1992. A simple, efficient PCR technique for
characterizing bacteriophage plaques. PCR Methods and Applications
2:93-95.

Hofmann, M. A. and D. A. Brian. 1991. Sequencing PCR DNA amplified directly
from a bacterial colony. BioTechniques 11:30-31.

Krishnan, B. R., R. W. Blakesley, and D. E. Berg. 1991. Linear amplification
DNA sequencing directly from single phage plaques and bacterial colonies.
Nucleic Acids Res 19(5):1153.

Krishnan, B. R., D. Kersulyte, I. Brikun, C. M. Berg, and D. E. Berg. 1991.
Direct and crossover PCR amplification to facilitate Tn5supF-based
sequencing of lambda phage clones. Nucleic Acids Res 19(22):6177-6182.

Krishnan, B. R., D. Kersulyte, I. Brikun, H. V. Huang, C. M. Berg, and
D. E. Berg. 1993. Transposon-Based and Polymerase Chain Reaction-Based
Sequencing of DNAs Cloned in lambda Phage. Methods in Enzymology 218:258-

================================================================================

25. What is solid-phase sequencing?
 
The use of a solid support for attachment of DNA has been used in order to
physically separate the single strands of DNA without the use of gel
purification. The opposite strands of DNA can then be independently sequenced
for verification. More recently, the use of paramagnetic particles has been
developed for use as the attachment medium. DNA strands can then be separated
by magnetic force and used in conjunction with sequencing techniques.

For more information see:

Fry, G., E. Lachenmeier, E. Mayrand, B. Giusti, J. Fisher, L. Johnston-Dow,
R. Cathcart, E. Finne, and L. Kilaas. 1992. A new approach to template
purification for sequencing applications using paramagnetic particles.
BioTechniques 13:124-131.
 
================================================================================
 
26. What is cycle sequencing?

Cycle sequencing is a technique that uses a thermal-cycling procedure similar
to PCR amplification for obtaining nucleotide sequence information from DNA
samples. The advantages of cycle-sequencing are that it is unnecessary to clone
a particular gene in order to get it's DNA sequence and that it requires very
little starting DNA material. The method is very similar to the standard
dideoxy sequencing, but uses the elements of PCR for amplifying the terminated
oligonucliotides used in the sequencing.

For more information see:

Rao, V. B., and N. B. Saunders. 1992. A rapid polymerase-chain-reaction-
directed sequencing strategy using a thermostable DNA polymerase from Thermus
flavus. Gene 113:17-23.

Ruano, G., and K. K. Kidd. 1991. Coupled amplification and sequencing of
genomic DNA. Proc. Natl. Acad. Sci. U.S.A. 88:2815-2819.

Smith, D. P., E. M. Johnstone, S. P. Little, and H. M. Hsiung. 1990. Direct
DNA sequencing of cDNA inserts from plaques using the linear polymerase chain
reaction. BioTechniques 9:48.

================================================================================

27. What is the easiest and most cost efficient means to remove the Dye
    Deoxy-terminators for automated sequencing after cycle sequencing?

The vast majority of fluorescent ddNTPs are not incorporated in the PCR products 
of cycle sequencing. If they are not removed effectively they form an enormous
peak at the start of a run and cause streak artifacts for several hundred bases
afterwards, thus seriously degrading the quality of the sequence.

The best way to remove the unincorporated dyes is to use a sephadex G-50
spin column. Make a hole in the bottom of 0.5 ml eppendorf tube using a hot 30
gauge needle and add about 25 ul volume of silanized zirconium glass beads.
Pour sephadex G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube. Place the
small eppendorf tube inside a larger 1.5 ml eppendorf tube and spin at about
500 rpm for couple of minutes to remove the excess liquid from the matrix.
Transfer the smaller tube to a clean siliconized 1.5 ml tube. The DNA sample
is added on top of the sephadex matrix and again spun at 1500 rpm for 2 minutes.
The eluate is either precipitated by at least 3 volumes of isopropanol or
dried in a roto-vac before loading. If you are able to see traces of remaining
dye within the cleaned sample, the sequence is usually so poor as to be unusable.

Another method relies on using Sephadex as a separation matrix, but in static
columns of pre-determined size. The problem of flow is overcome by having triton
X-100 in the mixture which does not affect the sequencing products.

The method is described in this paper:
 
Rosenthal, A., and D. S. Charnock-Jones. 1992. New Protocols for DNA sequencing
with Dye Terminators. Journal of DNA Sequencing and Mapping. 3:61-64.

================================================================================

28. Is there a sureshot method for double-stranded plasmid sequencing?



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