Team:British Columbia/Protocols/Site Directed Mutagenesis

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'''Site-Directed Mutagenesis with BioRAD iPROOF PCR kit'''
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<h1>Site-Directed Mutagenesis with BioRAD iPROOF PCR kit</h1>
   
   
This protocol closely follows the Stratagene QuikChange Site-Directed Mutagenesis kit, but uses the enzymes and buffers from the BioRAD iPROOF PCR kit.
This protocol closely follows the Stratagene QuikChange Site-Directed Mutagenesis kit, but uses the enzymes and buffers from the BioRAD iPROOF PCR kit.
   
   
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#Prepare the reaction mixtures
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:1. Prepare the reaction mixtures:
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#*Per 50 µL PCR reaction:
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{| class="wikitable"
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{| style="display: inline-table"
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|+Per 50 µL PCR reaction:
|-
|-
|dNTP||4 µL
|dNTP||4 µL
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|dH2O||to 50 µL
|dH2O||to 50 µL
|}  
|}  
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#*I would suggest trying 5 ng of dsDNA template, as I had the highest mutation success rate at this concentration.  This observation, however, has not been confirmed and is more anecdotal  
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::*I would suggest trying 5 ng of dsDNA template, as I had the highest mutation success rate at this concentration.  This observation, however, has not been confirmed and is more anecdotal  
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:2. Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length.
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#Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length.
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{| class="wikitable"
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1. Hot start 98 ºC 1:00
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|-
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2. Denaturation 98 ºC 0:10
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|1.||Hot start||98ºC||1:00
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3. Anneal 55 ºC 0:30
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|-
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4. Extension 72 ºC X:XX
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|2.||Denaturation||98ºC||0:10
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5. GOTO 2 for 12-18 cycles  
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|-
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|3.||Anneal||55ºC||0:30
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|-
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|4.||Extension||72ºC||X:XX
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|-
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|5.||GOTO 2 for 12-18 cycles  
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|}
The QuikChange protocol suggests the following number of cycles
The QuikChange protocol suggests the following number of cycles
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Type of mutation Number of cycles
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{| class="wikitable"
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Point mutations 12
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|-
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Single amino acid changes 16
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|<b>Type of mutation</b>||<b>Number of cycles</b>
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Multiple amino acid deletions or insertions 18
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|-
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|Point mutations||12
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3. Check for amplification on agarose gel
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|-
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NOTE: I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome
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|Single amino acid changes||16
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|-
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4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!)
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|Multiple amino acid deletions or insertions||18
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a. Add 10 U of DpnI to each reaction and incubate at 37 ºC
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|}
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:3. Check for amplification on agarose gel
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5. Transform 1 µL of PCR product.  I did not do any PCR purification before this step
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::*I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome.
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:4. Perform DpnI digestion of amplification products to digest the parental DNA ('''CRITICAL!!!''')
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:5. Add 10 U of DpnI to each reaction and incubate at 37 ºC
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:6. Transform 1 µL of PCR product.  I did not do any PCR purification before this step

Latest revision as of 01:35, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

Site-Directed Mutagenesis with BioRAD iPROOF PCR kit

This protocol closely follows the Stratagene QuikChange Site-Directed Mutagenesis kit, but uses the enzymes and buffers from the BioRAD iPROOF PCR kit.

1. Prepare the reaction mixtures:
Per 50 µL PCR reaction:
dNTP4 µL
iProof HF Buffer10 µL
MgCl21.5 µL
Primer, each? µL (125 ng)
dsDNA template? µL (5-50 ng)
iProof enzyme0.5 µL
dH2Oto 50 µL
  • I would suggest trying 5 ng of dsDNA template, as I had the highest mutation success rate at this concentration. This observation, however, has not been confirmed and is more anecdotal
2. Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length.
1.Hot start98ºC1:00
2.Denaturation98ºC0:10
3.Anneal55ºC0:30
4.Extension72ºCX:XX
5.GOTO 2 for 12-18 cycles

The QuikChange protocol suggests the following number of cycles

Type of mutationNumber of cycles
Point mutations12
Single amino acid changes16
Multiple amino acid deletions or insertions18
3. Check for amplification on agarose gel
  • I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome.
4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!!!)
5. Add 10 U of DpnI to each reaction and incubate at 37 ºC
6. Transform 1 µL of PCR product. I did not do any PCR purification before this step
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