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From 2012.igem.org

Contents 1 Mutagenesis 1.1 Prepare the sample reaction(s) as indicated below 1.2 Cycling Parameters for the QuikChange Lightning Site-Directed Mutagenesis Method 1.3 Loading sample 1.4 Injecting sample onto the column 1.5 Washing column

Mutagenesis

QuikChange® Lightning Site-Directed Mutagenesis Kit

Prepare the sample reaction(s) as indicated below

1. 5 μl of 10× reaction buffer
2. X μl (10–100 ng) of dsDNA template
3. X μl (125 ng) of oligonucleotide primer #1
4. X μl (125 ng) of oligonucleotide primer #2
5. 1 μl of dNTP mix
6. 1.5 μl of QuikSolution reagent
7. ddH2O to a final volume of 50 μl
8. Then add: 1 μl of QuikChange® Lightning Enzyme

Cycling Parameters for the QuikChange Lightning Site-Directed Mutagenesis Method

File:Mutagenesis thermal cyling
Figure 1:* For example, a 5-kb plasmid requires 2.5 minutes per cycle at 68°C.

Loading sample

1. Set flow rate on 0
2. Set loop valve on Load Position
3. Inject the sample into the loop with an syringe
4. Keep the syringe in the machine

Injecting sample onto the column

1. Set flow rate on 0
2. Set loop valve on Inject Position
3. Set column valve on designated position
4. Set on the pressure alarm: 5mPa
5. Set flow rate on half the maximum the column can handle
6. Run the FPLC for about 1 times the loop volume
7. Turn on the Frac 900, to collect the samples

Washing column

1. Set flow rate on 0
2. Empty waste bin
3. Set loop valve on Load Position
4. Set column valve on designated position
5. Set on the pressure alarm: 5mPa
6. Set flow rate on half the maximum the column can handle
7. Run the FPLC for about 2 times the column volume
8. Empty waste bin