Paul.xu at 16:21, 8 June 2020

2020-06-08T16:21:21Z

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== 2 X High Fiderity Polymerase PCR Mix from Thermofisher For Cloning ==

{| border="1" cellspacing="1" cellpadding="1"
|-
| 
| style="width: 193px;" | for 100 ul
|-
| H2O 
| style="width: 193px;" | 48 ul
|-
| 5 X  HiFi PCR Buffer 
| style="width: 193px;" | 40 ul
|-
| 10 mM dNTP
| style="width: 193px;" | 4 ul
|-
| 5 Units/ul of Superfi II from Thermofisher
| style="width: 193px;" | 4 ul
|}

== Mix, Spin briefly, Store at -20 oC ==

Use 5 ul for PCR, final volume is 10 ul

For 10 ul PCR

F primer: 0.5 ul

R Primer: 0.5 ul

templete, dip it; genomic DNA, 1 ul

2 X PCR mix: 5 ul

water: to 10 ul

== ==

== 2 X High Fiderity Polymerase PCR Mix For Cloning ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 691px;"
|-
| 
| 
| style="width: 193px;" | for 1 X 25 ul reaction
| style="width: 233px;" | for 10X 25 ul reactions
|-
| 10 mM dNTP
| 
| style="width: 193px;" | 1 ul
| style="width: 233px;" | 11 ul
|-
| 5 Units/ul of Q5, or Kfu, or Pfu or Kapa or Phusion
| 
| style="width: 193px;" | 0.5 ul
| style="width: 233px;" | 5.5 ul
|-
| 5 X  Q5 PCR Buffer
| 
| style="width: 193px;" | 5 ul
| style="width: 233px;" | 55 ul
|-
| or 10 X  HiFi PCR Buffer
| 
| style="width: 193px;" | or 2.5 ul
| style="width: 233px;" | or 27.5 ul
|-
| H2O
| 
| style="width: 193px;" |
6 ul if 5X buffer is used

8.5 ul if 10X Buffer is used

| style="width: 233px;" |
203.5 ul if 5X Buffer is used

231 ul if 10 X Buffer is used

|}

== 2X Taq PCR Mix for Analysis ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 691px;"
|-
| 
| 
| style="width: 193px;" | for 1 X 25 ul reaction
| style="width: 233px;" | for 10X 25 ul reactions
|-
| 10 mM dNTP
| 
| style="width: 193px;" | 1 ul
| style="width: 233px;" | 11 ul
|-
| 5 Units/ul of Taq
| 
| style="width: 193px;" | 0.5 ul
| style="width: 233px;" | 5.5 ul
|-
| 10 X Taq PCR Buffer
| 
| style="width: 193px;" | 2.5 ul
| style="width: 233px;" | or 27.5 ul
|-
| H2O
| 
| style="width: 193px;" | 8.5 ul
| style="width: 233px;" | 231 ul 
|}

== PT Mix (Primers + Template) ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 691px;"
|-
| 
| 
| style="width: 193px;" | for 1 X 25 ul reaction
| style="width: 233px;" | for 10X 25 ul reactions
|-
| 10 uM Forward Primer
| 
| style="width: 193px;" | 1 ul
| style="width: 233px;" | 11 ul
|-
| 10 uM Reverse Primer
| 
| style="width: 193px;" | 1 ul
| style="width: 233px;" | 11 ul
|-
| Optional: Template (miniprep DNA, or E coli culture)
| 
| style="width: 193px;" | 0.1~0.5 ul
| style="width: 233px;" | 0.1~0.5 ul
|-
| H2O
| 
| style="width: 193px;" | 10.5 ul
| style="width: 233px;" | 103 ul 
|}

On ice, aliquote 12.5 ul of 2 X PCR Mix to each PCR tube, add 12.5 ul PT Mix Add 0.1 to 0.5 ul of  template DNA sample to each tube if the PT Mix does not contain template. For example, E coli culture from colonies.
[[Category:Protocols]]

2X PCR Mix - Revision history

Paul.xu at 16:21, 8 June 2020