Dear researchers
In the process of optimizing the Tm of an amplification protocol and due to the lack of materials and reagents. Is it possible to reuse the PCR mix another time if the amplification program has failed? Knowing that in this mix there are the different components needed and the template DNA
Yousra Sbaoui It isn't recommended.
1) Because your dNTP's would have degraded after the thermal cycling. So they might not be able to give you amplification the next time.
2) You added polymerase the first time. Adding another round of fresh polymerase will mean the total glycerol % of the solution goes up. This may inhibit the reaction.
Might, I recommended you scale down the reaction instead? What total volume per PCR vial are you working with currently?
It is not a good idea because if this is a too high annealing temperature failure then one primer may have annealed and is increasing the amount of incorrect amplimer so I would expect the next lower temperature cycling to produce a dirty amplification but if you have never had a success with these primers then re running the assay at a much lower annealing temperature and adding half the original amount of Taq is probably worth a try. It is often easier to clean up a dirty amplification that has,at least, worked rather than a total failure of amplification which can be caused in many ways
If you are working with 50ul reaction volume then it may be a good idea to scale that down to about 20ul. Also, you could try doing a gradient PCR if you have the faculty to do so.
Thank you Henry Oamen Paul Rutland Rohit James for your answers. I use 20ul as final volume and I already tired gradient PCR. since it's not recommended so I'll avoid it and optimize the reagents
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