Muratore:Protocols/PCR/Quikchange: Difference between revisions
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(wrong volumes in table and added note about dNTPs) |
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==Basic QuikChange protocol== | |||
{|border="1" | {|border="1" | ||
|+ | |+ | ||
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|- | |- | ||
!Experimental | !Experimental | ||
| | |17 μL ||5 μL ||5 μL ||10 μL ||10 μL ||2 μL ||1 μL ||50 μL | ||
|- | |- | ||
!(-) Control | !(-) 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. | :* 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. | ||
Line 22: | Line 23: | ||
:# 15' @ 68°C | :# 15' @ 68°C | ||
:# hold @ 4°C | :# hold @ 4°C | ||
==2-stage QuikChange protocol== | |||
This protocol is based on the report by {{Ref|Wang & Malcolm (1999)}}. It is especially helpful for large inserts. | |||
{|border="1" | |||
|+ | |||
'''1st stage''' | |||
|- | |||
!Tube !!sterile H<sub>2</sub>O !!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: | |||
{|border="1" | |||
|+ | |||
'''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=== | |||
# {{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