I am working on a method where I need to amplify a short (150bp) sequence, and then I want to amplify that PCR product to increase concentration. The goal is to obtain a clear 150bp band after the second round of PCR.
After optimizing the first PCR, I was able to get a specific band. However, when I try to amplify that product, extra bands show up. I think there are two issues:
Attached is a gel where I ran negative controls. Note that the band is 200bp, so it is not the desired 150bp product. From the left, the lanes are:
So my questions are:
Dear, In second PCR how much PCR product used?
if you use a 1 ul or diluted 1st pcr product in 2nd PCR then you get a specific band.
hello
you can use this site ( sequence manipulation suite ) after design primer to check whether the primer contains a heterodimer or not
I don’t see why you surmise that primer dimer formation is an issue. The 200-bp band that you see in lane 2 (´both primers, no template ´´) must result from the amplification of some template DNA contaminating some of your reagents. You may get some information about what’s going on by sequencing the 200-bp species from both ends using the two primers
Hi Krishna Goyani, In the second PCR I use 1µL of a 1000x dilution of the first PCR product as template. Do you think that changing the concentration of the template to something else might help?
As Pierre Béguin said, this a clean reaction, either contamination mentioned or an indel within the amplicon you're amplifying. Is there a reason you don't run a large reaction or multiple reactions, combine them and use a PCR purification clean up kit to increa6sr the concentration?
You can reduce the amount of primers, this can help in primer dimer formation.
Always optimise your PCR primers (annealing temp, primer conc., and if not using a premade master mix then Mg conc. also). Avoid primers that are predicted to form primer-dimers - even though your product in lane 2 is not from primer-dimers (it's too big), significant primer-dimer formation reduces the availability of those primers for binding to your template. Are you using the same primers for step one and step two? Maybe consider a nested PCR design, where the first set of primers sit outside the amplicon region of your final product (dictated by your second set of primers).
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