Cloning parts by PCR using Kod polymerase
18 July 2008.
Kod Hot Start is a proof-reading polymerase distributed by Novagen. It is claimed to be as accurate as Pfu and 4 to 5 times faster. It comes with three solutions: 10 x reaction buffer, 2 mM dNTP mixture, and 25 mM MgSO4. These are stored in the 'PCR reagents' box in the freezer. before you start, take these out, thaw them, making sure they are completely thawed and well mixed before use, and store them on ice. Then, to a 0.5 ml PCR tube add:
- about 32 microlitres of water (adjust the amount to get 50 microlitres total volume at the end)
- 5 microlitres of 10 x reaction buffer
- 5 microlitres of 2 mM dNTP mix
- 3 microlitres of 25 mM MgSO4
- 1.5 microlitres of primer mixture (at 10 pmol/microlitre each)
- 1 to 2 microlitres of template (DNA or cell suspension)
- 1 microlitre of Kod polymerase (in the blue freezer box with the other enzymes).
To mix the reaction, set your P200 to 50 microlitres and pipette it gently up and down a few times, making sure that you don't introduce bubbles.
The cycling protocol is as follows: Program 1: initial denaturation at 95 C for 2 minutes. Links to program 3. Program 3: 30 cycles. Segment 1, 95 C for 20 seconds. Segment 2, annealing temperature for 10 seconds. Segment 3, extend at 70 C. Links to program 4. Program 4: chase at 70 C for 10 minutes, then cool to 4 C and hold.
The recommended extension time is 10 seconds/kb for fragments below 0.5 kb; 15 seconds/kb for fragments of 0.5 to 1 kb; 20 seconds/kb for fragments of 1 to 3 kb; and 25 seconds per kb for fragments of greater than 3 kb. (By comparison, Taq uses 1 minute/kb and Pfu 2 minutes/kb).
We have had good success with Kod Hot Start. The short extension time makes it ideal for fusion PCR, site-directed mutagenesis and other protocols where relatively long fragments (vector plus insert) are to be amplified.
Modification for high-GC templates
For high-GC templates or other difficult templates, we have found it advantageous to
- Add 10 microlitres of 50% v/v glycerol to the reaction mixture, decreasing the amount of water to maintain the same final volume. (We also tried DMSO, but glycerol seemed to work better.)
- Increase the length of the 95 C denaturation step in each cycle from 20 seconds to 1 minute.
In those cases where we have tried it, doing both of these things together works much better than just doing either one of them without the other. Theoretically, glycerol should decrease the strength of GC bonds (I think), so it might be necessary to use a slightly lower annealing temperature - we have not investigated this in any systematic way.