Hello everybody,
I would like to ask about one pair of my primers. Annealing temperature of my primers was calculated to 55°C. These primers give me product at annealing of 58°C with isolated bacterial DNA from LB medium. But, interestingly, with isolated bacterial DNA from DMEM F12 medium (yes, for mammalian cell culture medium, wo phenol red, see https://www.sigmaaldrich.com/catalog/product/sigma/d9785?lang=en®ion=CZ ) these primers give me product at annealing of 55°C. Could it be caused by the differences in both mediums, or what could be a cause of that?
Thanks a lot,
Pavlina
You do not say how much dna was amplified in each case. The efficiency of pcr depends partially on the amount of template dna present at the start of the reaction so if, for instance, you had less F12 dna it might take either more cycles or a decreased annealing temperature ( so a gteater pcr efficiency in the early cycles) to get the same amount of amplification between the 2 samples
Paul, thank you for your answer. DNA concentrations in both cases are same (50 ng/µl, according the manufacturer´s instruction for pcr reagents).
Dear Pavlína Nývltová , the annealing temperature of primer is directly dependent on the particular sequence of gene of organism rather than culture media from which it has been isolated. I think, there should not be any affect or difference in annealing temperature with the use of varying culture media.
Either you must have repeat the experiments using both of the DNA or you can put gradient PCR using range of annealing temperature from 52 to 60. Sometime, bands(with low or more intensity) usually seen in multiple annealing temperatures.
Thank you
Regards
Dear Amit, thank you for your answer. Unfortunately, we have already done gradient PCR (50-60°C), and F12 DNA gives product only at 58°C (2x) and LB DNA gives products up to 57°C.
Regards, Pavlina
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