Kyle’s protocol for long PCR
This protocol intended for generation of long PCR products (3-10kb) from mouse ES cell or tail DNA.
Template- If you want very long products, you musn’t shear the genomic DNA during preparation. A low salt prep will work best.
Polymerase- It is useful to make mixture of Taq and a high fidelity (proofreading) enzyme, typically Pfu, because the high fidelity enzyme remover misincorporated nucleotide that would otherwise be chain terminating. You can buy a prepared mixtures of Taq high fidelity enzymes from Perkin Elmer, Roche, or Stratagene, along with the buffer optimized for long products. However, in many cases it is sufficient to just use Taq (eg. “Red Taq” from Sigma) and its standard buffer, or else to mix Taq at a 30: 1 ratio of Taq: Pfu.
A good general buffer if you are forced to make it yourself is:
10X buffer 500 mM Tris pH 9.1 at 25C
160 mM (NH4)2 SO4
15 mM MG (OAc)2
Of course you may have to adjust the [Mg 2+] concentration upward to achieve better specificity. If the template has significant secondary structure, generating polymerase pause sites, then you can also include DMSO and b-mercapto ethanol in your 10X buffer. If there is any question, you should probably always use these, as they are unlikely to negatively affect your results in my experience.
The cycling profile should include a hot start. (ie no enzyme is added until the reactions have been at or above 80C for several minutes.) Duration time is critical, and should be no more than 20s at 94C. Ramp times should be rapid; hence a really old cycler might not be very useful. An annealing time of 40s is adequate in my experience. Extension times should be no more than appx. 1s/30nt, or around 33sec/kb. Specificity of the reaction might also be improved by extending at a lower than the normally prescribed temperature of 72C. Extension at either 68C or 70C works fine, and as mentioned above, is useful w/ long primers with melting temperatures in this range to rid oneself of a separate annealing step.
Given all the above, the following protocol was put together for creating a ~3.8kb PCR product including the entire IRS-1 coding region.
10mM dNTPS (2.5mM/ea) 2.5 0.25
DMSO 2.5 10%
b-mercaptoethanol 0.25 1%
total= 22ml+0.5-1u Red Taq/reaction
The cocktail is heated to>75C in a water bath for 5 minutes, then apportioned, 22ml each to the separate templates in their tubes.
NB: In tis case a principle was violated in that the primers were unmatched, having Tms of 72°C and 67°C. Both were 33mers. In general the most critical elements for success are buffers, the hot start, and a short denaturing time/proportional extension time.