Frequently Asked Question (FAQ) list for bionet.molbio.methds-reagnts

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Jun 8 17:23:38 EST 1993


*******************************************************************
*                                                                 *
*                Frequently Asked Question (FAQ) list             *
*                for bionet.molbio.methds-reagnts                 *
*                                                                 *
*                Last update was on 08 June 1993                  *
*                                                                 *
*******************************************************************

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.

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 for anonymous FTP from
net.bio.net [134.172.2.69] in pub/BIOSCI/biosci.FAQ. It may also be
requested by sending e-mail to biosci at net.bio.net (use plain English
for your request). The FAQ is also posted on the first of each month
to the newsgroup BIONEWS/bionet.announce immediately following the
posting of the BIOSCI information sheet.

Another good source of information for new users is "A Biologist's
Guide to the Internet" which is published monthly in the Usenet
newsgroups sci.bio, bionet.general, and news.answers. It is also
available by anonymous ftp from pit-manager.mit.edu as file "biology/guide".

This FAQ list for bionet.molbio.methds-reagnts is available by anonymous
ftp from ncifcrf.gov as the file pub/methods/FAQlist. It is also available
by anonymous ftp from net.bio.net as pub/BIOSCI/METHDS-REAGNTS/METHODS.FAQ

ftp archives of all the BIOSCI/bionet messages are available at net.bio.net 
[134.172.2.69] in /pub/BIOSCI. Contact biosci at net.bio.net for further help
or comments on the archives.

If anyone would like to make additions or corrections to this FAQ list,
please send the information to:

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
e-mail: pnh at ncifcrf.gov 

================================================================================
 1. What are some good reference books for methods in molecular biology?
 2. Where can I get information about the genotypes and phenotypes of
    common E. coli strains used in molecular genetics?
 3. How can I access MEDLINE?
 4. How do I amplify pBR322 plasmid using chloramphenicol?
 5. 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?
 6. How I do prepare powdered silica for DNA purification, with the
    associated solutions?
 7. How do I use powdered silica to isolate DNA from agarose gels?
 8. How do I use silica powder to prepare plasmid DNA for sequencing?
 9. What is PCR?
10. What are some good reference books for PCR?
11. How should I select a set of primers to use for PCR?
12. What kinds of programs are available for designing PCR primers?
13. What is "Hot-start" PCR?
14. What is AP-PCR or RAPD PCR?
15. What is "Touchdown" PCR?
16. Is there a simple method to sequence lambda, M13, or plasmid
    clones using PCR?
17. What is solid-phase sequencing?
18. What is cycle sequencing?
19. What is the easiest and most cost efficient means to remove the Dye
    Deoxy-terminators for automated sequencing after cycle sequencing?
20. Is it possible to clean and re-use electroporation cuvettes?
21. Is there a simple subcloning method for plasmid construction?
================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

1. 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.

Promega Corporation provides a detailed manual for many procedures
called "Promega Protocols and Applications Guide". To get one contact:

Promega Corporation
2800 Woods Hollow Road
Madison, Wisconsin 53711-5399 USA

Toll free: 1-800-356-9526
phone: 608-274-4330
FAX: 608-273-6867
Telex: 62057092

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

2. Where can I get information about the genotypes and phenotypes of common
   E. coli 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

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

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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

3. How can I access MEDLINE?

First, you'll need to get an account on MEDLARS. Call the MEDLARS Management
Section Service Desk: 800-638-8480. 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.

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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

4. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

5. 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        20        30        40        50        60        70        80 

6. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

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

To purify DNA from agarose gel, weigh 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

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

This method 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

9. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

10. What are some good reference books for PCR?

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

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.

This book contains selected articles on sequencing PCR products published
in the journal BioTechniques up until March 1992:

Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing.
Eaton Publishing Co., Natick, Mass.

Here is an extensive review article with 236 references regarding PCR:

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.

Another general paper on PCR is:

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).

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

11. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

12. 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 WREngels at wisc.macc.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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

13. 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:

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

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

14. 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-

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

15. 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-

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

16. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

17. 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.
 
================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 
 
18. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

19. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 
 
20. 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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 

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

Low-melt agarose method:

Cut your vector and insert fragments and run them out on a low melting agarose
gel (eg. SeaKem).  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 eppendorf
tubes. Add 5 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:

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.

================================================================================
.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80 
<end of file>



More information about the Methods mailing list