Frequently Asked Questions (FAQ) List

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Sat Nov 7 16:20:21 EST 1992


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*                Frequently Asked Question (FAQ) list             *
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*                Last update was on 01 November 1992              *
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Many people on the net have contributed to the construction
of this question-and-answer format. Since I have completely
lost track of how many people have helped form the answers,
I would just like to thank all those who have helped in one
way or another. I hope that the FAQ list will continue to grow
and others will also help keep the information up to date.

This FAQ list is available by anonymous ftp to ncifcrf.gov
Simply get the file pub/methods/FAQlist

If anyone would like other methods to be available within this
ftp site, I will gladly add them here.

Also, if anyone would like to make additions or corrections to
the 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 

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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.

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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 papers provide detailed descriptions of many E. coli strains:

Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia
coli K-12. Bacteriol. Rev. 36:525-557.

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

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. Each 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 USA 20852 USA
phone (301) 231-5585

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.

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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

If you haven't got telnet, you can access MEDLINE via BITNIS and get your
MEDLINE searches returned via email. You can do searches in command format or
by using Grateful Med 6.9 and a program called smed.

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.

You can access MEDLINE on the Internet through a service called the Life 
Science Network.  This is a database service that offers access to over
80 life science databases, including MEDLINE.  Although you will need a
password and User ID to access the service, there is no charge to sign up
and you're only charged for the information you retrieve -- no 
monthly minimum fees.  To get a User Agreement, e-mail to:

biosis at a1.relay.upenn.edu.

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.

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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.

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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.

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How do I calibrate a Pipetman(tm) style micropipette?

To clean the inside of the pipettor:

Pull off the tip ejector and then unscrew the barrel. Inside you will
find a stainless steel plunger assembly (which differs slightly in
each model), a small rubber O-ring and a small teflon seal. Be very
careful when handling these as they are easy to damage and easy to lose.

Once apart clean the plunger and inspect the O-ring and seal. Also
clean out the barrel, making sure that the surface where the O-ring
seals against the barrel is clean. If the teflon seal or O-ring is
damaged, they will need to be replaced. If your pipettes "drip", it
is likely that the seals are the problem. If the steel plunger is
extremely pitted from corrosion then you are in trouble. In all
likelihood your pipettes are filthy inside and a good cleaning will
probably fix the problem.

By disassembling and cleaning your pipettes once a month or so you can
prevent damage to the seals and plunger and avoid the need for costly
recalibration or early replacement.

If the seal is OK and it is still pipetting inaccurately, it may need
recalibrating. Actually this is a misnomer, since the "calibration" of
the pipetman is fixed by the pitch of the micrometer threads and can
never be "out of calibration". What can change and be changed is the
limit on the upper end of the travel of the plunger (the one with the
button on it that your thumb touches). The other thing that can be
changed (by mistake) is the proper remounting of the pair of knurled
knobs that flank the rubber "Friction ring" - but only if it was
removed to replace the friction ring (which crack and fall off after
a few years - another worthwhile spare part to get.)

Determine the error of the pipettman at each of three or four settings
equally spaced across the range it is capable of measuring.
Recalibration is called for if the error is constant (or nearly so)
in volume. 

To calibrate the pipettor:

First determine if you have the old style pipettor or the new style.
If the pipetter has hex nuts on the adjustment dial then you have an
older one that can be calibrated if necessary. For the older models,
an allen wrench and a screw driver are all that is needed.

The newer ones take a "forked" tool that fits into the two holes on
the top of the adjustment dial - that tool is very expensive.
Gilson sells the special tool for adjusting the calibration on these.
You don't need to buy their exorbitantly-priced special tool though.
You can use a tool with plier-like handles and blunt pins on the end
of the jaws that can be bought at a local discount department store.
It is also possible to use the metal wire from a disassembled small or
medium sized binder clip (the same type as used for sequencing gels
but smaller). You can use the end to insert into the hole of the inner
ring on top of the adjustable dial. Hold the inner ring still using
the clip while turning the outer dial. This will change the calibration set.

Recalibration (changing the limit on the upper taravel of the shaft)
is done by using an allen (hex head) wrench to loosen the three or
four set screws in the TOP knurled knob (above the friction ring).
Once loose, a small flat end screw driver can be used to move the
metallic colored slotted collar around the shaft. It is threaded and
has a very fine pitch, so more than one turn may be needed. With the
proper tool this wouild be easier to do. With each adjustment, try out
the pipetman again to see if the correct setting was obtained.

To test it, weigh pipetted distilled water on the analytical
balance. Water used as a standard should provide a good measure as to
where to set the loosened thumb screws. Remember 1.0 ul should weigh
1.0 mg. When obtained, tighten the set screws and check the
calibration at each of several volumes, depending on what volume
you normally pipet with that particular tool. Checking at 50% and 100%
of the total volume will work just fine for a P1000, but the P20
pipetman can be off by a significant amount depending on where you've
done the calibration, ie. at the 1 - 5 ul mark or the 15 - 20 ul mark.
Try to calibrate a P20 near the level of normal use. For example, if
you always use your P20 for measuring 1 - 5 ul, calibrate it at 3 ul.
This is important if you measure such small volumes on a regular
basis. If you calibrate at 20 ul, it may be off at the lower volumes.

If you need help, call Rainin (if in the US) for tech help over the
phone. Rainin (Gilson) pipettors go back to Rainin at Mack Road, Woburn,
Massachusetts USA. They will clean and recalibrate them for you, BUT the
price is almost as much as a total replacement (cost= $100 and up).
You can also buy a self-calibration kit from Gilson.

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Is it possible to get a personal copy of the files stored at Genbank?

NCBI has announced a CDrom based genbank distribution for cheap.. $50-$60
The package, called entrez, contains the mac and pc search software.  

Ask for a mailing from NCBI (202) 738-3238 (the fax number for orders 
is 202 512-2233)

Or contact Dennis Benson via Email (benson at ncbi.nlm.nih.gov)
 
NCBI also offers a retrieve service via email that is more up to date than
a CDrom could ever be.  It's a little clunky now, but improving.  For help,
Email retrieve at ncbi.nlm.nih.gov with the word <help> in the body of the message.

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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.

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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.

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

This method is based on Morelle's modification of the Birnboim and Doly 
alkaline lysis method. See:

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.

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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.

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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).

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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.

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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 anonymous ftp at 36.45.0.126 or contact
James Mullins jmullins.stanford.edu (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.

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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.
 
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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-

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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-

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