KOD PCR

Fill an ice bucket and get the KOD MM (hot start) from -30 freezer. Thaw the MM in 37℃ water bath and vortex. Collect all other necessary reagents (primers, DNA, etc.) and vortex before adding them together. Label PCR tubes.

Procedure for 50 uL reaction with two primers (Mainly used for plasmid DNA. For full length KOD protocol, click here):

Add the following:
 * 25 uL KOD MM
 * 1.5 uL 10 uM primer 1
 * 1.5uL 10 uM primer 2
 * Calculated amount (uL) needed for 10 ng of plasmid (Ex. If you have 2.4 ng/uL of DNA, you would need about 4.167 uL of DNA to achieve 10 ng of plasmid.)
 * Calculated autoclaved H20 to reach 50 uL volume

KOD Program:
 * 1) Polymerase Activation: 95 ℃ for 2 minutes
 * 2) Denature: 95 ℃ for 20 seconds
 * 3) Annealing: Lowest Primer Tm ℃  for 10 seconds
 * 4) Extension: <500 bp = 70 ℃  for 10 seconds/kb; 500-1000 bp = 70℃  for 15 seconds/kb; 1000-3000 bp = 70℃ for 20 seconds/kb; >3000 bp = 70℃ for 20 seconds/kb
 * 5) Repeat steps 2-4: 20-40 cycles