Try raising the temp for amplification by 2 degrees. If this causes
both to give amplification, it suggests that the temperature
differentiation of the primer's upper level 61.7 vs 61.0 is the
critical difference: the .7 degrees difference may be the influencing
factor that compensates for the low amplfication temperature.
If not: the significance is suspected to be the lower temperature
variation of set two, which disables the compensation for the low
amplficiation temperature.
This is just a thougt. Try it, if you can afford it.
Dave
Wellcome Trust Research Labs, Nairobi wrote:
>> To the news group
>> I am amplifying 2 fragments of an unique DNA sequence of malaria;
> pfmdr1 gene with 2 different sets of primers. All primers are 21
> nucleotides length .Set1 primers have Tm of 59 and 61 °C and Set2 have
> 61.7 and 51.2°C.
> PCR is carried out in standard condition and the Tm=40 °C. Using
> a same amount of DNA template (5 ul) of purified culture DNA, only Set2
> give an amplification. Set1 give an amplification if the template is 20
> to 50 times more. How can someone explain this difference in the PCR
> efficiency. The gene target is the same only the site of primers
> annealing differs. Moreover, Tm primers are quite similar and I used very
> low Tm (40 °C) for amplification.
> So, when the amount of DNA is not high (naturally infected
> sample), Set2 can not amplify the gene however Set1 does. Is there a
> problem in my PCR condition? As the PCR conditions are the same for the 2
> sets, why this difference?
>> Alexis
>> FOR ANSWER: PLEASE PRECISE MESSAGE FOR ALEXIS NZILA
