I have been working on site-directed mutagenesis for over 1 month, but still haven't been able to get mutant colony. I used Pfu High-Fidelity DNA polymerase to amplify my vector with insert and saw target band after the PCR. I added 1µl DpnI to 25 µL PCR reaction, incubation 37C, overnight. I purified the PCR product by PCR purification/clean up kit and measured the amount of DNA by NanoDrop (usually 20-30ng/µL). Then, I used all volume or half of purified PCR product for transformtion to 100µL competent DH5a. After overnight incubation at 37°C, I had several colonies. The positive control (transformed with native plasmid) had a lot of colonies and negative control (without transformation) had not colony, so I think my competent cells work well. I extracted the plasmid of some colonies, digested the plasmids with restriction enzyme but I don’t saw the bands of digested plasmid on gel. I don’t know why I don’t have the mutant colony. If you have any suggestion, please help me. Thank you so much.
It is possible that your mutation is presence, but your enzyme is dead. Have you verified that your restriction enzyme is active? Also, how big of a fragment are you expecting?
I suggest you follow the quick change protocol (if indeed thats the kit you are using) and check the samples they recommend on an agarose gel. That will tell you whats working. Make sure your oligo sequences are correct (and screen them against your entire clone not just the gene you want to mutate on a computer. Use only yhte nucleotide on the 3' end of the mutation and see if there is another binding site). You can check your DpnI separately - why incubate overnight? Its only 1 hour in the protocol and no PCR product purification. After extracting plasmid from colonies recovered we sequence 5 - usually at least 4 have the mutation we intended. It only takes a morning to make a mutant these days. If in doubt buy a new kit - its worth the money.
First confirm the mutation by sequence analysis.
The major point as you mentioned " I purified the PCR product by PCR purification/clean up kit and measured the amount of DNA by NanoDrop (usually 20-30ng/µL"
It seems to be low try to get the adequate amount of concentration after purification.
The another problem might be during transformation. The electroporation may not be successful.
If electroporation is successful then you can run the digestion for long time to get the band.
Take precautions during ligation and check by gel electrophoresis at every step.
If still problem persist I would like to advise you to elaborate your question
Vector details, restriction sites, size of the gene and vector.
hope it will help you.
Best of luck
When we use QuickChange, we never bother doing PCR cleanup. If you get a strong band of the correct size on your gel, your DpnI digest should be enough.
If you're still having trouble with QuickChange, you may want to try overlap extension PCR instead. We have had great success with this technique. We use universal primers outside our gene and then cut and ligate the product back into the original vector. As with other methods, you will want to sequence your products to make sure everything is ok.
Article Gene splicing and mutagenesis by PCR-driven overlap extension
For the good quality of the mutagenesis, the oligos have to be of high purity (PAGE quality). After your PCR check your reaction with agarose gel. After DPnI treatment you don't need to do PCR purification.
good luck
I once experienced the same problem (no colonies), I tried PCR-purification of the PCR-product even after dpnI-digest using a commerical kit and this lead to success. Maybe there is a component in your PCR-buffer that kills off the cells.
@Peter Rehbein
as I understood, Asma got colonies after transformation, but those appear to be not-mutated.
It can be that the DpnI enzyme doesn't work and therefore you have colonies that are not positive.
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