Hello,
I have a question regarding PCR. I am trying to do a site-directed mutagenesis on my plasmid.
I use fresh PCR water, change tips after adding something into one tube and I also let all the components thaw on ice before using them. I first add PCR water, then my plasmid template, then the primers and at last the MasterMix. I tried different annealing temperatures but only one time it worked. After repetition the band was lost again. Still my band is missing and I am quite confused what the reason is.
You can only see one band at a different bp-amount on the gel and also see some band for primer dimers quite faintly.
What could I be missing? What could I change?
Hi Sarah Mattern
You should give attention to certain things.
- Before adding to PCR mixture, mix the components properly by short vortexing followed by a quick spin down. Sometimes thawing is not enough for proper mixing of the mastermix, primers, and the template. Especially the salt in the mastermix is precipitated after thawing on ice until mixed properly.
- Run a temperature gradient PCR with different annealing temperatures and standardize the optimum annealing temperature.
- If you have long primers or your primers tend to form secondary structures, then add 1.1M betaine to the mixture. It reduces the annealing temperature and the formation of secondary structure.
- Check the extension time of the PCR. Set a few seconds higher time than the polymerase extension ability. If the PCR product is too long (>2kb), the extension is slower than the shorter product.
All the best.
I used to do SDM for plasmids as well. A few things you can try in addition to the ones suggested above
run small volume reactions (20 ul or so) rather than a larger one (my original protocol was for 50 ul). You can do replicate tubes to get enough product
try a different polymerase, I had success with Vent from New England Biolabs
add some Magnesium, it can help in the same general way as Betaine and it might be something you already have around in the lab
Hello! I'd be happy to help. Could you provide me with more information about your PCR setup and the specific issue you are experiencing?
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU