I am working with two different genes of whitefly at the same time. Earlier both of the genes were successfully amplified in desired size. All the PCR components and temperature conditions are same except the primers but only one gene is getting amplified and not the other one. I have tried four or five different companies' PCR kits along with high fidelity taq, Phusion master mix etc. I have performed PCR more than 55 times trying different components, temperature conditions but till now I am not getting the full length gene.
Suggestions are invited.
Thank You.
Hey
Do you know the sequences where the primers align and the primers' sequences?
Maybe they don't fit because there are some point mutations.
Kind regards,
Adrian
Greetings,
What do you mean by different primers? Are they different in sequence? There are some sequences that does'nt seem to Like to be amplyfied by a simple PCR, in those cases you can make use of nested PCR technique. You need a set of 3 primers (FWD1, FWD2 and RV1). The first pair is used to amplify some kind of large gene region. Once the first run is done, take the PCR product as template for a second run, using FWD2 and RV1, you have to be sure that FWD2 corresponds to a sequence inside the amplicon generated by FW1 and RV1. Also, as Adrian says, your primer aligning region might be polymorphic, in that case I'd recommend you to use a low aligning temperature. You could try running a temperature gradient PCR from 45 to 50 °C to see if this solves your problem.
Best regards
Hi there
- If both primers previously worked then obviously there is not a problem with your primers
- Thus, using different kits etc was probably futile
- It could be the actual cDNA sample and/or stringency of the reaction
- How many times has you current cDNA aliquot been freeze thawed? Is it more than 3?
- Did the genes previously amplify from another batch of cDNA?
- Are your annealing conditions optimal in general for robust amplification
- I would suggest re making a couple of cDNA aliquots and then performing an annealing gradient with 'affected primer' wherein you vary the annealing temp from the Tm to Tm-5C in 1C intervals; in other words 5 x PCR reactions where you drop the annealing temp successively by 1C, viz Tm, Tm-1, Tm-2...Tm-5
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