Team:British Columbia/Protocols/Site Directed Mutagenesis
From 2012.igem.org
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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 | ::*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. | |
{| 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 | + | |Type of mutation||Number of cycles |
- | Multiple amino acid deletions or insertions 18 | + | |- |
+ | |Point mutations||12 | ||
+ | |- | ||
+ | |Single amino acid changes||16 | ||
+ | |- | ||
+ | |Multiple amino acid deletions or insertions||18 | ||
+ | |} | ||
:3. Check for amplification on agarose gel | :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. | ::*I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome. |
Revision as of 01:31, 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