Polymerase QC

1. Measure protein concentration of enzyme stock using nanodrop.

2. Make serial dilutions using enzyme storage buffer

Final Conc	Dilution Tube #	concentration	Dilution
Original	1	x mg/ml
1 ug/ul (1 mg/ml)	2	take5 ul #1 ---> dilute to x *  5 ul with storage buffer for example take 5 ul of 5 mg/ml, add 20 ul of storage buffer (storage buffer has 50% glycerol)	1
100 ng/ul	3	5 ul of #2, add 45 ul of storage buffer	1:10
50 ng/ul	4	2.5 ul of #2, add 47.5 ul of storage buffer	1:50
10 ng/ul	5	5 ul of #3, add 45 ul of storage buffer	1:100
5 ng/ul	6	5 ul of #4, add 45 ul of storage buffer	1:500
1 ng/ul	7	5 ul of #5, add 45 ul of storage buffer	1:1000

3, PCR

PCR Mix for 5 samples + 1 positive control

Components	1 reaction (25 ul)	for 6 Reactions	for 9 Reactions
10 mM dNTP	1 ul	7 ul	10 ul
5X Q5 PCR Buffer	5 ul	35 ul	50 ul
10 uM Forward	1 ul	7ul	10 ul
10 uM Reverse	1 ul	7ul	10 ul
H2O	16.5 ul	16.5 * 7=115.5 ul	165 ul
template (plasmid DNA)	0.1 ul	0.6 ul	1 ul

For Aliquot 25 ul of PCR Mix to PCR tubes. Put on ice, 

+ Control: Add 0.5 ul of active DNA polymerase (commercial or home-made, such as Q5 or Phusion) to  the last tube.

Add 0.5 ul of serial diluted enzyme to the rest of tubes.

Place tubes on PCR machine.

Run PCR, annealing temp is 55 ^oC, extension time is 1 kb/min.

KOD: ~ 19,384 U/mg, 19 U/ug, 5 Units/263 ng http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0131757

KOD Buffer: 10 mM Tris-HCl, pH 8.8, 1mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.01% Triton-100, and 0.1 mg/ml bovine serum albumin 10X Buffer: 100 mM Tris-HCl, pH 8.8, 10 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 0.1% Triton-100, and 1 mg/ml bovine serum albumin