Site Directed Mutagenesis
This protocol was drafted by Jill-Heidinger Pauli (2011) and subsequently modified by Vinny Guacci and Thomas Eng.
1. Dilute Qiagen mini-prep 1:2 using 1x EB (~ 75ng to 100 ng/PCR rxn)
2. Dilute each primer to be used to 2uM using H2O (2ul 100uM + 98ul H2O).
3. Make cocktail as for each plasmid to be mutated as shown below.
23 µL 2x Q5 Master Mix
23 µL ddH20
1 µL plasmid DNA (~ 100ng = 1:2 dil)
47 µL total volume
4. Place 22.5 ul cocktail mix into e/ RXN w/ (R) anti-sense]
To tube #1 add: 2.5 ul F primer (2uM stock = 0.2uM final)
To tube #2 add: 2.5 ul R primer (2uM stock = 0.2uM final)
6. Run PCR program, SEVEN CYCLES
segment cycles temp time
1 1 98°C 3 min
2 7 98°C 15 sec
60°C 30 sec
68°C ____ min*
3 1 4°C <10 min
* time used is 1 min/per plasmid size (kb) so use 9.5min for 9.5kb plasmid.
The ramp speed must be set to 1°C/sec
7. You need to be at the PCR soon after the reactions cools to 4°C (~ 30min). Open tubes & mix together the 25ul reactions for primers F & R, then resplit back to 25ul in the original tubes.
6. Run 12 cycles more for 19 cycles total.
segment cycles temp time
4 12 98°C 15 sec
60°C 50 sec
68°C ____ min* (same time as above)
5 1 68°C 7min
6 1 15°C whenever
7. Pool PCR then add 1ul DpnI to 50ul pooled PCR reaction and incubate 1hr at 37°C.
8. Bacterial transformation: E coli XL1 0.5 ul or 3 uL DpnI digested DNA (<1/10 DNA/cell vol) then plate entire amount onto one LB+KAN/etc plate. Should see between 20-100 colonies on the plate at 30˚C overnight. If no colonies, try incubating plates at 37 or 23 as well (and check thermostat in your warm room)