Team:British Columbia/Protocols/Site Directed Mutagenesis
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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. | ||
- | 1. Prepare the reaction mixtures: | + | :1. Prepare the reaction mixtures: |
{| class="wikitable" | {| class="wikitable" | ||
|+Per 50 µL PCR reaction: | |+Per 50 µL PCR reaction: | ||
Line 41: | Line 42: | ||
|dH2O||to 50 µL | |dH2O||to 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 | + | ::*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. | |
- | 2. Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length. | + | |
- | + | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
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|} | |} | ||
The QuikChange protocol suggests the following number of cycles | The QuikChange protocol suggests the following number of cycles | ||
- | Type of mutation Number of cycles | + | {| class="wikitable" |
- | Point mutations 12 | + | |- |
- | Single amino acid changes 16 | + | |<b>Type of mutation</b>||<b>Number of cycles</b> |
- | Multiple amino acid deletions or insertions 18 | + | |- |
- | + | |Point mutations||12 | |
- | 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 | + | |Single amino acid changes||16 |
- | + | |- | |
- | 4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!) | + | |Multiple amino acid deletions or insertions||18 |
- | + | |} | |
- | + | :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 |
Latest revision as of 01:35, 4 October 2012
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:
dNTP | 4 µL |
iProof HF Buffer | 10 µL |
MgCl2 | 1.5 µL |
Primer, each | ? µL (125 ng) |
dsDNA template | ? µL (5-50 ng) |
iProof enzyme | 0.5 µL |
dH2O | to 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 start | 98ºC | 1:00 |
2. | Denaturation | 98ºC | 0:10 |
3. | Anneal | 55ºC | 0:30 |
4. | Extension | 72ºC | X:XX |
5. | GOTO 2 for 12-18 cycles |
The QuikChange protocol suggests the following number of cycles
Type of mutation | Number of cycles |
Point mutations | 12 |
Single amino acid changes | 16 |
Multiple amino acid deletions or insertions | 18 |
- 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