We have a very robust, one-oligo, method for quick-change mutagenesis.
We routinely perform 24, 48, or 96 reactions for our customers. We pick 4 colonies from each sample, then miniprep for sequencing in 96-well plates.
If your looking to make larger insertions or deletions, we have found that a SLIC method gives a higher success rate.
Quick Change Mutagenesis Protocol
Design one forward primer to anneal at 55C both upstream and downstream of the point mutation (approximately 45bp total primer length). Use the primer design spreadsheet if you need it. Change the fewest number of bases possible to get the desired amino acid.
|4||Plasmid (160ng total)|
|2||Pfu Ultra buffer|
* NOTE: It is very important to use Pfu Ultra, NOT Pfu UltraII!!
|68C||2min/kb up to 20min|
|2||NEB Buffer 4|
Incubate at 37c for 2.5-3 hours.
Anneal & Transform
1. Add 6ul to 100ul of competent cells on ice
2. Incubate on ice for 30min
3. Heat shock at 42C for 30sec
4. Immediately place on ice for 2min
5. Add 900ul 2YT and shake for 1hr at 37C
6. Plate 150ul on appropriate antibiotic plate
For a 96-well project, use the spreadsheet below to easily design your primers.
96 QC Primer Worksheet
You can also download the protocol below.