This is the protocol for site-directed mutagenesis based on the Stratagene kit.
- Pfu turbo
- 10X Pfu turbo buffer
- dNTPs (10mM)
- Forward and reverse primers (0.1ug/uL, see methods section for design tips)
- competent cells
- Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this
- Primers should have a minimum GC content of 40% and terminate in one or more C's or G's
- Tm should be greater or equal to 78 degrees Celsius and can be calculated as follows:
- Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch
Set up as follows:
|Template DNA (50ng/uL)||1 uL|
|10X Buffer||5 uL|
|Forward Primer (0.1 ug/uL)||1 uL|
|Reverse Primer (0.1 ug/uL)||1 uL|
|dNTPs (10mM)||1 uL|
|Pfu turbo||1 uL|
Depending on the length of the plasmid, this program can become very long, so it may be best to run overnight.
- 95 degrees for 1 minute
- 95 degrees for 50 seconds, 60 degrees for 50 seconds, 68 degrees for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total
- 68 degrees for 7 minutes
- 4 degree hold
Add 1uL of Dpn1 to PCR reaction. Incubate at 37 degrees Celsius for 1-2 hours to digest parental DNA. Run 5uL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern. Transform into competent cells.