Tips for increasing your PCR specificity (decrease nonspecific product formation)

[@thebumblingbiochemist]

We said that higher is better in terms of specificity, which is true. But too high is worse in terms of efficiency and yield. Remember that above the Tm, most copies of the primer won’t be bound. So there’s a sort of best-of-both-worlds strategy you can try called Touchdown PCR. It starts with a cycle or two at a high anneal temp, so you give the most specific products an (exponential) head start and then you decrease the anneal temp in subsequent cycles so you can increase yield - with copies coming from those original good copies that will swamp out any subsequent bad ones. Another temperature-based strategy to try is using HotStart Polymerases. These have something that makes them inactive (such as an antibody bound to them) until you raise the temperature in that initial melt step and that blocking thing falls off and/or gets inactivated, etc. more on this here:   bit.ly/hotstartPCR & kzbin.info/www/bejne/o6TLhZ6Kbb5-iq8 But the purpose of it is that it prevents the polymerase from doing any copying while you are setting up your reaction and you have lower temperatures, potentially higher concentrations of primers, etc. Which brings me to another thing you can optimize to improve specificity… *decrease template and/or primer concentration to reduce risk of nonspecific product formation The more wrong sites there are for the primer to find, the more likely you’ll get bad products. And the more primer copies there are, the more chances there are for mispriming to occur. High primer concentrations also raise the risk of primer dimer-related problems. Finally (though probably not last in terms of order of testing since it’s simple) you can try adding an additive like DMSO which can decrease secondary structure in your template, help the template strands melt, and thus make it easier for the primer copies to find the target you want. This is especially important if the template region containing that target is GC-rich. There are also specific buffers you can use with GC-rich sequences. I recommend optimizing for specificity and then, if you need a higher yield, perform multiple PCR reactions and pool them together during purification - you can prepare a scaled-up reaction in one tube but then split it down to the volume you optimized at for the actual running (i.e. don't try to run it on the large volume). More on this here: blog: bit.ly/pcryield ; KZbin: kzbin.info/www/bejne/eGSkmXmsnb6BoZI   Hope that helps! Promega has a nice guide on optimizing your PCR: www.promega.com/resources/guides/nucleic-acid-analysis/pcr-amplification/#general-considerations-for-pcr-optimization-6f575242-fe99-4fcb-88c5-b205ad7becc7 And here are some links to other posts of mine PCR playlist: kzbin.info/aero/PLUWsCDtjESrHbBVTt6wqWBGGbd0Q9pbRA more on PCR: bit.ly/pcrtrain & kzbin.info/www/bejne/fYu2fZl7eMyKabM   more on nucleic acid spin columns: bit.ly/spincolumns & kzbin.info/www/bejne/nKuVgKOgrbZ-ga8     more on agarose gel electrophoresis: bit.ly/agarosegelcompare & kzbin.info/www/bejne/rJPYqZlpp8iArsk     more practical lab tips: bit.ly/lab_tricks_page & kzbin.info/aero/PLUWsCDtjESrFEAWZCRKJL7sMc6a_KgfLU    more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@ylidene6222]

Thank you for your videos, you help many people who feel lonely in this path

[@thebumblingbiochemist]

So happy I could help and so sorry you feel lonely

[@haribhaskaran8607]

Great video for troubleshooting your PCR! Thank you

[@thebumblingbiochemist]

Thanks! Happy to help

[@guacamole8554]

Thank you 🙏

[@cezreycor]

I recommend a smaller glove size. other than that, great vid! you're amazing! :) thanks!

[@thebumblingbiochemist]

Thanks so much - so glad you found it helpful!