Hello, everyone, I want to create 6 SDM at a different location in the coding region using 6Kb Plasmid. I had chosen 3 different sites among 6 sites and started to mutate using QuikChange II XL Site-Directed Mutagenesis Kit.
I used below mention PCR (18 cycles) program I got the colonies.But unfortunately, all colonies are wild type even I changed the site of mutations but still, all are wild type.
94 for 3'
94 for 1'
52 for 1'
68 for 6'
68 for 60'
4 for hold
Step2---Dpn-1 digestion at 37 degrees for 1hr.
Step3--- Purification of PCR product using the phenol-chloroform method.
Step4 -Transform all volume approx's (8-10ul) in 50ul DH5alpha.
Step5-Saturate the transformant for 1hr at 37 degrees.
Step6-- Plate on LBA with Amp+ incubate O/N.
Step7-- Inoculation in LB with Apm+ for O/N.
After sequencing, all are wild type.
Could any one share an idea to do 6 SDM one after other?
As Iris said, check if you have PCR product. Assuming your DpnI digestions worked and you see lots (how many?) WT colonies suggests that PCR worked.
Are you generating all 6 mutants in one PCR or one by one? How far are the mutants? (In other words, how big is the primer?) generating 6 mutant at a time will be difficult possibly due to non-specific primer binding).
Check your PCR primers again if they correctly have mutated nucleotide (I know, but if something doesn't work then you have to start from bottom).
In PCR cycle, 68 for 60 minutes seems unusual? why that long, 5-10 min will be sufficient.
Thanks
If you hardly see any PCR amplification, modify your primer design as described https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91, this modified method proved much more efficient than the others.
Hi.. Actually, my object to create the 6 mutations in the single gene one by one and Now I'm struggling only to getting the 1st one.If I will get one mutation the I used the mutated plasmid as Template for next round of mutation. Their location between 15-50 Nts to each other. And my primers are--as follows 1st to 6th-32,29,32,25,31,40.
And I used the PCR program going through is article--https://www.ncbi.nlm.nih.gov/pmc/articles/PMC514394/pdf/gnh110.pdf
Thank you
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