Muratore:Protocols/PCR/Quikchange

From OpenWetWare

< Muratore:Protocols | PCR(Difference between revisions)
Jump to: navigation, search
(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) ...)
Current revision (16:05, 23 June 2011) (view source)
 
(5 intermediate revisions not shown.)
Line 1: Line 1:
 +
==Basic QuikChange protocol==
{|border="1"
{|border="1"
|+
|+
Line 8: Line 9:
|-
|-
!Experimental
!Experimental
-
|12 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||1 μL ||50 μL
+
|17 μL ||5 μL ||5 μL ||10 μL ||10 μL ||2 μL ||1 μL ||50 μL
|-
|-
!(-) Control
!(-) Control
-
|13 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||0 μL ||50 μL
+
|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.
-
# 30" @ 95°C
+
:* 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
+
:# 30" @ 95°C
-
# 8.5' @ 68°C
+
:# 1' @ 55°C
-
# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
+
:# 1'/kb @ 68°C
-
# 10' @ 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 {{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]]

Current revision

Basic QuikChange protocol

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.
  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

2-stage QuikChange protocol

This protocol is based on the report by Wang & Malcolm (1999). It is especially helpful for large inserts.

1st stage
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.
  1. 30" @ 95°C
  2. 30" @ 95°C
  3. 1' @ 55°C
  4. 1'/kb @ 68°C
  5. remove from PCR machine, pipet off wax, and chill briefly to re-harden thin wax layer
  6. go immediately to 2nd stage:
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.
  1. 30" @ 95°C
  2. 30" @ 95°C
  3. 1' @ 55°C
  4. 1'/kb @ 68°C
  5. repeat steps 2-4 x 17 more times
  6. 15' @ 68°C
  7. hold @ 4°C

References

  1. Wang & Malcolm (1999) - Biotechniques 26 680
Personal tools