Editing Polymerase QC

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;" | <br/>
|-
| style="width: 84px;" | 
1 ug/ul

(1 mg/ml)

| style="width: 81px;" | 2
| style="width: 283px;" | 
take5&nbsp;ul #1 ---> dilute to x *&nbsp; 5 ul with storage buffer

for example

take 5&nbsp;ul of 5&nbsp;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&nbsp;of storage buffer
| style="width: 58px;" | 1:10
|-
| style="width: 84px;" | 50 ng/ul
| style="width: 81px;" | 4
| style="width: 283px;" | <span style="color:#FF0000;">2.5 </span>ul of #2, add <span style="color:#FF0000;">47.5</span> ul&nbsp;of storage buffer<br/>
| 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<br/>
| 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<br/>
| 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<br/>
| style="width: 58px;" | 1:1000
|}

3, PCR

PCR Mix for&nbsp;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&nbsp;ul
|-
| 5X Q5 PCR Buffer&nbsp;
| 5 ul
| 35 ul
| 50&nbsp;ul
|-
| 10 uM Forward
| 1 ul
| 7ul
| 10&nbsp;ul
|-
| 10 uM Reverse
| 1 ul
| 7ul
| 10&nbsp;ul
|-
| H<sub><sup>2</sup></sub>O
| 16.5&nbsp;ul
| 16.5 * 7=115.5 ul
| 165&nbsp;ul
|-
| template (plasmid DNA)
| 0.1 ul
| 0.6 ul
| 1&nbsp;ul
|}

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

<span style="color:#FF0000;">+ Control:</span> Add 0.5 ul of active DNA polymerase (commercial or home-made, such as Q5 or Phusion) to&nbsp; 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 <sup>o</sup>C, 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]]