Advice for PCR: NZILA ALEXIS

Wellcome Trust Research Labs, Nairobi wellcome at USERS.AFRICAONLINE.CO.KE
Fri Jan 24 04:23:43 EST 1997


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



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