I have been working on site-directed mutagenesis for a few months now, without success. I used a High-Fidelity DNA polymerase to amplify my vector with an insert and saw the target band after the PCR when I ran the product on a gel. I added 0.5µl DpnI to my 50 µL PCR reaction, and incubate at 37°C for an hour. Then, I used 25ul of the PCR product for transformation to 100µL competent XL1-blue cells. After overnight incubation at 37°C, I had got no mutant colonies. The positive control had a lot of colonies and the negative control (without transformation) had no colony, so I think my competent cells work well.
Additionally, I had previously done this work and had managed to get colonies but this time around I am not getting anything at all.
Anyone with expertise on this, may I please get some assistance? Thank you so much.
Dear Thando Kay
1) was the band of the PCR at the expected size? Since sometimes, if your primers are not really specific you can amplify smaller fragments of the vector that miss some parts essential for vector replication or antibiotic resistance and
2) Did you try to repeat the trasformation with lower amout of PCR? 25ul are a lot, some times also too much DNA can be toxic for the cells and also the buffers of PCR and dpnI can affect the trasfromation. In general, if the band is visible in agarose gel 1-2ul are more than enough.
3) can you share with us the sequence of the primers that you used for perform the PCR, hust to do a double check of the lenght and orientation of the overlapping regions.
However for the future i would like to suggest you to use the PIPE cloning approach, which do not require any specific kit, but just an high fidelity taq polimerase and thermo MACh1 competent cells.
you can find more information about it on the following paper
https://pubmed.ncbi.nlm.nih.gov/18988020/
of in the following links
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
and
https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html
good luck
Manuele
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