Muratore:Protocols/PCR/Quikchange: Difference between revisions
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(wrong volumes in table and added note about dNTPs) |
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!Experimental | !Experimental | ||
|12 μL || | |12 μL ||5 μL ||5 μL ||10 μL ||10 μL ||2 μL ||1 μL ||50 μL | ||
|- | |- | ||
!(-) Control | !(-) Control | ||
|13 μL || | |13 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. | ||
:* dNTPs should be aliquoted to minimize freeze-thaw cycles and ensure the integrity of the triphosphate. | |||
:# 30" @ 95°C | :# 30" @ 95°C | ||
:# 30" @ 95°C | :# 30" @ 95°C |
Revision as of 08:33, 9 June 2011
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 | 12 μL | 5 μL | 5 μL | 10 μL | 10 μL | 2 μL | 1 μL | 50 μL |
(-) Control | 13 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