Muratore:Protocols/PCR/Quikchange

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(wrong volumes in table and added note about dNTPs)
Line 8: Line 8:
|-
|-
!Experimental
!Experimental
-
|12 μL ||10 μL ||5 μL ||10 μL ||10 μL ||2 μL ||1 μL ||50 μL
+
|12 μL ||5 μL ||5 μL ||10 μL ||10 μL ||2 μL ||1 μL ||50 μL
|-
|-
!(-) Control
!(-) Control
-
|13 μL ||10 μL ||5 μL ||10 μL ||10 μL ||2 μL ||0 μL ||50 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 10:33, 9 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 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.
  1. 30" @ 95°C
  2. 30" @ 95°C
  3. 1' @ 55°C
  4. 1'/kb @ 68°C
  5. repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
  6. 15' @ 68°C
  7. hold @ 4°C
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