Dear Researchers,
I am finding difficulty in amplifying opaque-2 gene using gene specific SSR marker phi057. After PCR when I run electrophoresis I will be left only with smear and no bands are visible. I tried even gradient PCR but then the bands are lightly visible with so much of smear.
My PCR conditions are
940C for 5 mins----- Initial denaturation
940C for 45 secs ------ denaturation
560C for 45 secs------ aneealing 35 cycles
720C for 1 min------- extension
720C for 10 mins---- Final extension
I usually go for 10microliter reaction
Hi Anitha,
There must be some problem with the PCR per se...If there are no reports of PCR amplification of this gene...you may need to optimize it a little bit more..You have mentioned only about gradient PCR.... no other information is provided about the size of the amplicon...and reaction components and conditions..It is likely the smear can be due to any of the factor...you need to check these one by one...and try to put a +ve control also to make sure things are working fine...Increasing the volume may also help
bye
Hi,
You should check primers (specificity, number of match and mismatch nucleotides for Tm calculation), integrity and purity of template DNA, concentration of primers and template and softly pipet your PCR components and then mix them. PCR volume does not have an effect on PCR product, but the concentration of PCR component strongly affects it.
be helpful
please post your gel picture...
is your ladder is also giving smear?
Good afternoon, dear P. Shetti.
The times that I saw smears in samples were due to, basically, poor quality of the DNA sample and also to unspecific amplification (Wrong annealing temperature). A long time of denaturation could also affect the efficiency of amplifcation but since this is not the case, it doesn't apply to your situation.
First, as colleagues recommended, you should re-check your quality control parameters: Purity, Integrity, Concentration. Second, consider your Tm temperatures, if even doing a gradient PCR doesn't help, you could go for touchdown PCR (High temperatures to maximize specificty and then lower temperatures to increase yield), but this will depend on what your future purposes are for these samples.
There are some details that could contribute to optimize your current protocol too: I strongly recommend you to try it out with 20ul-PCR mix reaction, then you could economize the reagents by reducing to 12.5ul.
Always check the "nature" of the desirable region to amplify, consider GC content or if it's a promoter region. I had to deal with a SNP located in a promote region and it wasn't until we realized about that that we increased the denaturation time and use 95°C instead of 94°C.
Please, feel free to talk with me, It would be a pleasure to figure it out how to fix this trouble,
Best regards,
Brayan
A little bit smear is too common in PCR products with SSR markers, but if it's so much based on my own experience, it's better you increase annealing temperature,
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