Muratore:Protocols/PCR/Quikchange: Difference between revisions
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==2-stage QuikChange protocol== | ==2-stage QuikChange protocol== | ||
This protocol is based on the report by Wang & Malcolm | This protocol is based on the report by {{Ref|Wang & Malcolm (1999)}}. It is especially helpful for large inserts. | ||
{|border="1" | {|border="1" | ||
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:# 15' @ 68°C | :# 15' @ 68°C | ||
:# hold @ 4°C | :# hold @ 4°C | ||
===References=== | |||
# {{FormatRef|Wang & Malcolm|1999| |Biotechniques 26|680| }} | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
[[Category:PCR]] | [[Category:PCR]] |
Latest revision as of 14:05, 23 June 2011
Basic QuikChange protocol
Tube | sterile H2O | Pfu Buffer | Template | For primer | Rev primer | dNTPs | Pfu Turbo | wax |
---|---|---|---|---|---|---|---|---|
(10X) | (10 μg/mL) | (12.5 ng/μL) | (12.5 ng/μL) | (10 mM ea) | (2.5 U/μL) | |||
Experimental | 17 μL | 5 μL | 5 μL | 10 μL | 10 μL | 2 μL | 1 μL | 50 μL |
(-) Control | 18 μL | 5 μL | 5 μL | 10 μL | 10 μL | 2 μL | 0 μL | 50 μL |
- Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
- dNTPs should be aliquoted to minimize freeze-thaw cycles and ensure the integrity of the triphosphate.
- 30" @ 95°C
- 30" @ 95°C
- 1' @ 55°C
- 1'/kb @ 68°C
- repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
- 15' @ 68°C
- hold @ 4°C
2-stage QuikChange protocol
This protocol is based on the report by Wang & Malcolm (1999). It is especially helpful for large inserts.
Tube | sterile H2O | Pfu Buffer | Template | For primer | Rev primer | dNTPs | Pfu Turbo | wax |
---|---|---|---|---|---|---|---|---|
(10X) | (10 μg/mL) | (12.5 ng/μL) | (12.5 ng/μL) | (10 mM ea) | (2.5 U/μL) | |||
Experimental-forward | 27 μL | 5 μL | 5 μL | 10 μL | 0 μL | 2 μL | 1 μL | 50 μL |
Experimental-reverse | 27 μL | 5 μL | 5 μL | 0 μL | 10 μL | 2 μL | 1 μL | 50 μL |
(-) Control-forward | 28 μL | 5 μL | 5 μL | 10 μL | 0 μL | 2 μL | 0 μL | 50 μL |
(-) Control-reverse | 28 μL | 5 μL | 5 μL | 0 μL | 10 μL | 2 μL | 0 μL | 50 μL |
- Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
- dNTPs should be aliquoted to minimize freeze-thaw cycles and ensure the integrity of the triphosphate.
- 30" @ 95°C
- 30" @ 95°C
- 1' @ 55°C
- 1'/kb @ 68°C
- remove from PCR machine, pipet off wax, and chill briefly to re-harden thin wax layer
- go immediately to 2nd stage:
Tube | 1st stage-forward | 1st stage-reverse | Pfu Turbo | wax |
---|---|---|---|---|
(2.5 U/μL) | ||||
Experimental | 25 μL | 25 μL | 1 μL | 50 μL |
(-) Control | 25 μL | 25 μL | 0 μL | 50 μL |
- Mix DNA and Pfu by pipetting, then add wax.
- 30" @ 95°C
- 30" @ 95°C
- 1' @ 55°C
- 1'/kb @ 68°C
- repeat steps 2-4 x 17 more times
- 15' @ 68°C
- hold @ 4°C
References
- Wang & Malcolm (1999) - Biotechniques 26 680