Team:Wageningen UR/Protocol/Mutagenesis
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Revision as of 10:59, 26 October 2012
Contents |
Mutagenesis
QuikChange® Lightning Site-Directed Mutagenesis Kit
Prepare the sample reaction(s) as indicated below
- 5 μl of 10× reaction buffer
- X μl (10–100 ng) of dsDNA template
- X μl (125 ng) of oligonucleotide primer #1
- X μl (125 ng) of oligonucleotide primer #2
- 1 μl of dNTP mix
- 1.5 μl of QuikSolution reagent
- ddH2O to a final volume of 50 μl
- Then add: 1 μl of QuikChange® Lightning Enzyme
Cycling Parameters for the QuikChange Lightning Site-Directed Mutagenesis Method
Loading sample
- Set flow rate on 0
- Set loop valve on Load Position
- Inject the sample into the loop with an syringe
- Keep the syringe in the machine
Injecting sample onto the column
- Set flow rate on 0
- Set loop valve on Inject Position
- Set column valve on designated position
- Set on the pressure alarm: 5mPa
- Set flow rate on half the maximum the column can handle
- Run the FPLC for about 1 times the loop volume
- Turn on the Frac 900, to collect the samples
Washing column
- Set flow rate on 0
- Empty waste bin
- Set loop valve on Load Position
- Set column valve on designated position
- Set on the pressure alarm: 5mPa
- Set flow rate on half the maximum the column can handle
- Run the FPLC for about 2 times the column volume
- Empty waste bin