Paul.xu at 00:13, 9 February 2018

2018-02-09T00:13:53Z

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1. Measure protein concentration of enzyme stock using nanodrop.

2. Make serial dilutions using enzyme storage buffer

{| border="1" cellspacing="1" cellpadding="1" style="width: 528px;"
|-
| style="width: 84px;" | Final Conc
| style="width: 81px;" | Dilution Tube #
| style="width: 283px;" | concentration
| style="width: 58px;" | Dilution
|-
| style="width: 84px;" | Original
| style="width: 81px;" | 1
| style="width: 283px;" | x mg/ml
| style="width: 58px;" | 
|-
| style="width: 84px;" |
1 ug/ul

(1 mg/ml)

| style="width: 81px;" | 2
| style="width: 283px;" |
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)

| style="width: 58px;" | 1
|-
| style="width: 84px;" | 100 ng/ul
| style="width: 81px;" | 3
| style="width: 283px;" | 5 ul of #2, add 45 ul of storage buffer
| style="width: 58px;" | 1:10
|-
| style="width: 84px;" | 50 ng/ul
| style="width: 81px;" | 4
| style="width: 283px;" | 2.5 ul of #2, add 47.5 ul of storage buffer 
| style="width: 58px;" | 1:50
|-
| style="width: 84px;" | 10 ng/ul
| style="width: 81px;" | 5
| style="width: 283px;" | 5 ul of #3, add 45 ul of storage buffer 
| style="width: 58px;" | 1:100
|-
| style="width: 84px;" | 5 ng/ul
| style="width: 81px;" | 6
| style="width: 283px;" | 5 ul of #4, add 45 ul of storage buffer 
| style="width: 58px;" | 1:500
|-
| style="width: 84px;" | 1 ng/ul
| style="width: 81px;" | 7
| style="width: 283px;" | 5 ul of #5, add 45 ul of storage buffer 
| style="width: 58px;" | 1:1000
|}

3, PCR

PCR Mix for 5 samples + 1 positive control

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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 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
[[Category:Protocols]]

Polymerase QC - Revision history

Paul.xu at 00:13, 9 February 2018