Hi,
I´m working on site-directed mutagenesis trying to mutate several short stretches of ~6-7 nt within a 5 kbp plasmid. I designed mutagenesis primers. In 2 PCR reactions the 2 DNA fragments are generated which are fused in the last reaction (classical mutagenesis PCR). After restriction digest of insert and vector, vector dephosphorylation, vector-insert-ligation and heat shock transformation I checked on colonies and isolated the plasmid but the mutation was never incorporated. Although I get a PCR product all the time I fear that the proofreading DNA Pol (Vent) may have reversed the mutation due to my non-specific PCR conditions. All PCR products are gel extracted and spin column purified prior to further processing. I think my plasmid DNA template was isolated from E. coli Top 10. Is DpnI digestion necessary although all PCR products are gel extracted? And why I do not get any mutations because I performed everything 4 weeks ago without gel extraction of products and got 1 positive in 3 picked colonies.
Thanks so much for any suggestion!
The PCR reaction which I use (in general):
a.95°C 3 `
b.95°C 30´´
c.Lowest annealing temp. which is used for a primer pair...30´´ (I try to save time and run single reactions in a 8er-stripe (which I believe may be the problem)
d. 72°C 1min/kb
b.-d. 30 cycles
72°C 5´
Hi, Stefan,
You used overlapping PCR to generate mutants. It is not necessary to digest the original plasmid DNA with DpnI. If you design your mutant in two overlapping primers (forward and reverse), and amplify the entire plasmid (your mutant in the new plasmid), you need to digest the old (original) plasmid by DpnI first then transform into E. coli.
Chunhong
I agree with Chunhong that with overlap PCR you shouldn't need to get rid of the original plasmid with DpnI.
However, perhaps the most likely option is that something is wrong with your ligation step. Sometimes dephosphorylation isn't efficient enough. Did you try gel purifying your digested vector to make sure no insert was left to religate (in case dephosphorylation is incomplete)? How many different ratios of insert:vector did you try?
If the restriction site is directly at the end of the PCR product, restriction enzymes can have difficulty functioning properly. Did each of your PCR primers include additional nucleotides at the 5' end to allow for efficient restriction digestion? NEB recommends adding 6nts 5' of the recognition site in each primer, although you can look up how well your enzymes of interest do with different lengths: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments.
If you're still stuck I'll try to think of something else.
Hi Amanda, Hi Chunhong,
Thanks so much for your quick answers.
I think I need to specify even more what I´m trying to do. Actually I try to mutate a short sequence in a 3´UTR which itself is inserted in a Luciferase Reporter vector. This 3´UTR sequence has previously been cloned into this vector so restriction sites are present. So I do the Mutation on the Basis of this sequence present in the vector. Gel purification is done for PCr products and the digested vector in parallel. In primer design I usually add 2 more nucleotides at the beginning of the restriction site.
Someone suggested in Troubleshooting for site-directed mutagenesis to abolish the final 72°C 5 min Extension step during PCR as this will reduce the risk of reversal of the Mutation? Would you agree on this?
So, I noticed that DpnI Digestion is not necessary in my case....
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