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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: | |
| - | + | {| class="wikitable" | |
| - | {| | + | |+Per 50 µL PCR reaction: |
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|dNTP||4 µL | |dNTP||4 µL | ||
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|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 | |
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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. | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | |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 | The QuikChange protocol suggests the following number of cycles | ||
Type of mutation Number of cycles | Type of mutation Number of cycles | ||
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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 | |
4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!) | 4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!) | ||
Revision as of 20:20, 22 June 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!) a. Add 10 U of DpnI to each reaction and incubate at 37 ºC
5. Transform 1 µL of PCR product. I did not do any PCR purification before this step
