issue with PCR - (Nov/11/2018 )

I need some help with a PCR. I am amplifying cDNA that was converted from RNA of a tissue section. I have three donors. I am targeting a specific gene and used different annealing temperatures.

The first time I carried out the PCR, everything worked at one of the annealing temperatures. I got the right bands, then cloned, grew colonies on agar plates, and Sanger sequenced for verification of target and it was verified.

I repeated the cloning and agar plate colony growth to get more colonies. The second time I didn't see the same PCR bands at the same annealing temperature. Instead I saw the correct bands at different annealing temperatures. I thought it was strange, but I cleaned the product up (PCR purified) and again streaked agar plates for colonies and Sanger sequencing. I saw colonies on all the plates, colony picked them by restreaking on a new agar plate, then submitted them out for Sanger sequencing. The vendor said there was no colonies that grew.

My question are

-what protocol do you recommend for cDNA amplification of gene targets to detect isoforms? Do I have to plate them for colony picking after PCR? Is it necessary to do a PCR cleanup?

-My PCR bands were very faint both times, what polymerases do you recommend for this type of workflow?

-what part of the steps I wrote above would you troubleshoot?

Hi Biogirl,

There are some steps missing in your description that make it a little hard to answer. What kind of vector plasmid did you clone your PCR products into?  Was it something that makes background colonies very rare so that all the colonies that grow must contain insert? Is it a ligase or topoisomerase kit? It seems like you just sent colonies off to be sequenced, so the vendor does the plasmid preps for you? Do you not make plasmid preps and confirm and insert by restriction digestion first? I’m not familiar with kind of workflow.

It sounds like your PCRs are working- to get a greater yield you might try adding a few more amplification cycles to your rxn. Just 3-5 cycles, don’t overdo it.  It’s OK if they work at different annealing temperatures as long as you get what you want. What polymerase kit I would chose would depend on what my downstream application was (e.g. clone for a construct, just get the sequence, or just test presence/absence of product), what my cloning vector is, and how long the PCR products were supposed to be. What kit or kind or polymerase are you using? How big are your bands?

Is your goal to just get the sequence of the PCR products or do you want to do something more with the cloned fragments afterward? If it is the first option, you do not need to clone the products or put them into bacteria to sequence them.  You can submit PCR products for Sanger sequencing but yes, you do have to clean them up to get rid of leftover amplification primers, nucleotides, etc.  Then you can sequence them with ONE of the amp primers per sequencing rxn. You can have two reactions done, one with your forward primer and one with the reverse primer. Granted you will get neater sequencing results, and a better read of the very ends of the fragments, if you clone them into a vector first.