(Fwd) Advice for PCR: NZILA ALEXIS

Xinzhuan Su xsu at HELIX.NIH.GOV
Fri Jan 24 09:03:57 EST 1997


>------- Forwarded Message Follows -------
>To:            parasitology at net.bio.net
>From:          wellcome at USERS.AFRICAONLINE.CO.KE ("Wellcome Trust Research
>Labs, Nairobi")
>Subject:       Advice for PCR: NZILA ALEXIS
>Date:          24 Jan 1997 01:23:43 -0800
>
>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

Dear Alexis,

Obviously the differences are the primers. Amplification efficiency is
affected by the efficiency of primers annealing to the template. I think
the best way is to play with different annealing temperatures. If the GC
content of your primers are higher than 50%, I think you should try 50-60
C. Another way is to make another pair of primers (if 20-mer with 6-8 CG,
annealing Temp. should be around 48-54C).

The size of the product will affect the yield too. The larger, the lower
yield (most cases). If you are amplifying more than 3 kb, try Strategene's
taqplus with low salt buffer. Generally, taqplus give higher yield on all
PCR products.

If there is an 'intron' region (high AT) in your product, you should run
your PCR with extension temp. at 60 C (see Nucleic Acid Research
V24:1574-75).

I don't understand your 'low Tm (40C)'. Is that your annealing temp.? If
so, it may be too low, try 48-52C. If you are not sure of annealing temp.
try a cycle with: 94C, 20 sec; 48C, 10 sec; 52C, 10 sec; and 60c, ? sec.

Hope these will answer your questions and help to improve your PCR yield,
and GOOD LUCK!







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