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)