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Long PCR Protocol

Long PCR Reagents and Guidelines

(Modified from Cheng et al. (1))


General Guidelines for Long PCR Conditions and Enzyme Mixtures

Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others. If you are interested in using other polymerases make sure that you use compatible buffers. Error rates for other polymerases can be found at http://research.nwfsc.noaa.gov/protocols/taq-errors.html

For PCR with low-complexity templates (e.g., plasmid and cosmid inserts)

50 microliter reaction in 1X Long PCR buffer (5X recipe below)

For PCR with moderate-complexity templates (e.g., bacterial genomic DNA)

For PCR with high-complexity templates (e.g., human genomic DNA)


Long PCR Buffer

In our hands tricine buffer works well with Tth but not as well with Taq. The pH is probably critical to the efficiency of amplification of long targets (1).

1X Long PCR Buffer

A 5X buffer stock containing 6 mM Mg(OAc)2 (1.2 mM final concentration) and 5% DMSO (1% final concentration) can easily be made using 500 mM Mg(OAc)2, 1 M Tricine and 1 M KOAc stock solutions as follows:

5X Long PCR Buffer

10 ml 5X buffer


Cycle times and temperatures

Generally, we have been using two temperature cycles with one annealing/extension step @ 68 degrees C and a short melting step @ 94 degrees C.Presently, a rough formula for calculating annealing/extension times is 1 min + (2.5 sec/100 bases) = n. The constant one minute is probably necessary for primer annealing/extension to occur; at 68 degrees C the kinetics of primer-template annealing and melting may become the limiting factor in the rate of primer extension.

Generic Long PCR Program for the Perkin Elmer 9600

The 15 sec cycle extension for cycles 16-30 may be necessary for only the longest PCR (20 kb), please experiment.

Hot Starts

I use a hot start method for all of my L-PCR. I split the reaction into two parts: a template/primer fraction which is 3/4 or 4/5 of the reaction volume, and a polymerase fraction which constitutes the remaining 1/4 or 1/5 of the reaction. Each fraction is 1X for buffer concentration. The polymerase fraction contains only polymerase, buffer and water; all other components are included in the template/primer fraction. I put the template/primer fraction in the tube and heat in the PCR machine to 94 degrees C for 10 sec. to denature. I then add the polymerase fraction during the first annealing/extension step. Alternatively, after denaturing, an 80 degree step can be used for adding the polymerase fraction(P.E.).


Picking Primers

We have had success using the following guidelines for primers (these are not inviolable rules, they are simply guidelines):

REFERENCES

1. Cheng, S., Fockler, C., Barnes, W., Higuchi, R. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. 91, 5695-5699 (1994).


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Last updated 2 June 2000 by Pete Estep, pestep@rascal.med.harvard.edu