I am using 0.2mM dNTPs,40pm primers,0.6microlitre Taq,2 microlitre MgCl2(2.5mM) in a total 20 microlitre reaction volume for PCR. I have kept the temperature the same so far. I need a band size of 850 bp. But using the recipe mentioned, I am getting just 600bp band. Please can someone guide me so that I can 850bp?
Maybe the annealing temperature of the primers isn't ideal and thus its efficiency is compromised and the desired size is not amplified.
Hi Usman,
I would like to clarify some aspects. If you are getting 600bp band then the question is, is the band non-specific? The question I ask, because a 600 bp non-specific band means two things: First Primer sequence is not unique and it's matching at some place else, giving rise to specificity. The second is if it's primer dimer. The second question gets nullified because to think of a dimer of 600bp match is quite obnoxious thought. So, I shall recommend you to double check the primer. Rest aside the Tm. It is also important, but the primer Seq in such an observation is more important.
If the primer is tested and tried, may be with help of an earlier referent work, then I would suggest you see whether this band comes in No Template Control (NTC). If band comes in NTC at the abberant 600bp mark, then I would suggest that the primers/reagents are contaminated. Hence, you have to change each in batches. Even if that does not work, then I think your annealing and expansion time should be made longer with a much stringent condition, like increase the MgCl2 condition.
These are some tested and triad aspects I have done. Hope this will help you.
Best,
Somnath Paul.
What comes immediately to my mind when I hear about too short products are two things: Which polymerase are you using and how long is your amplification time? Are you sure that your primers are correct and only bind at the desired places?
Did you checked if the primers will give the proper band size? are you targeting against chromosome or plasmid? Your question is somehow so vague that it is hard to answer.
As most people have mentioned you might be getting the correct product for the primers you have designed, it would be difficult to think of a dimer of 600bp.I would start by reviewing the primers (are the primers specific ? . I addition to what has been mentioned you need to look into your template material, is this happening on one sample or several samples ? Is it the region prome to deletions ?
Check your overall PCR optimisation first, then focus on the primers you are using because unless you have some form of contamination, my first guess would be that you primers are non-specific. You could maybe try to use primers with a high Tm maybe above 60 to minimise the undesired product. Let us know when you figure it out.
If the 600 bp band appears like a smear, also consider that your input DNA could be partially degraded, resulting in products with smaller size.
Somethin that jumped at me: you're using 0.6 ul of Taq for a 20 ul reaction. That seemed a little excessive for me, most PCR mix reactions used 1 ul Taq for a 50 ul reaction. Maybe you could double-check your Taq concentration and recommended reaction mixture set up. Although excessive Taq is likely not a factor for our current problem, it just seems quite wasteful to me considering that Taq typically is the most expensive reagent in a PCR mixture.
check your primers for nonspecific binding
U can try to do insilico pcr an find the band size.
Also make sure you have the right DNA ladder. Confirm the band sizes in the ladder and then compare.
@ Artur Burzynski
Thanks for your comment!
@ Usman Ali
Why don't you ask your supervisor first?
Verifica que los primers diseñados si amplifiquen el tamaño que esperas, haz un BLAST colocando el forward y el reverse separados por 10 espacios contra la base de todos los nucleotidos. Una vez hayas confirmado que si amplifican los nucleotidos esperados, simplemente revisa el tiempo que le estas dando al termociclador en la extensión. Así que ensaya dejar la extensión en 1 min a 1:30 min en cada ciclo y una extensión final de 10 min.
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