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