Difference between revisions of "Muratore:Protocols/PCR/Quikchange"

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(New page: {|border="1" |+ '''Basic QuikChange protocol''' |- !Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax |- ! !! !!(10X) !!(10 μg/mL) ...)
 
Line 13: Line 13:
 
|13 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||0 μL ||50 μL
 
|13 μL ||10 μL ||10 μL ||10 μL ||5 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.
# 30" @ 95°C
+
:# 30" @ 95°C
# 30" @ 95°C
+
:# 30" @ 95°C
# 1' @ 55°C
+
:# 1' @ 55°C
# 8.5' @ 68°C
+
:# 8.5' @ 68°C
# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
+
:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
# 10' @ 68°C
+
:# 10' @ 68°C
  
 
[[Category:Protocol]]
 
[[Category:Protocol]]
 
[[Category:DNA]]
 
[[Category:DNA]]
 
[[Category:PCR]]
 
[[Category:PCR]]

Revision as of 10:03, 26 May 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 10 μL 10 μL 10 μL 5 μL 2 μL 1 μL 50 μL
(-) Control 13 μL 10 μL 10 μL 10 μL 5 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.
  1. 30" @ 95°C
  2. 30" @ 95°C
  3. 1' @ 55°C
  4. 8.5' @ 68°C
  5. repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
  6. 10' @ 68°C