Problems with RT-PCR

Patrick HJ Falckh p.falckh at rmit.edu.au
Tue Jan 9 19:38:44 EST 1996


karbach at wbzx06.biozentrum.uni-wuerzburg.de (Doris Karbach) wrote:
>Dear Netters,
>I am doing quite a lot of RT-PCR and I was sure to convince all initial
>problems. Now I want to detect the mRNA of one protein, which I could detect
>rather good in Western Blots. I am sure that my RNA is not degraded and
>Primers fit very well. I have raised the concentration of MgCl2 and it went
>only slightly better. Has anyone an idea what to make better
>Thanks in advance Doris

If you have only tried a small increase in [Mg++] then I assume that you haven't optimised the reaction relative to Mg++, annealing temp or cycle number.  I've found that the standard range of Mg++ does not always cover the optimal range of the primers, no-matter how much computer time I spend ensuring that the reaction will work.  Additionally I had an experience where the plastic I was using was a chelator of the Mg++ and I needed to raise the final conc. to 5.5mM to be optimal !!
There are a number of kits on the market that can 'speed-up' the optimisation process, however, you can make your own up after that.

In Erlich's book "PCR Technology" he shows an example of primer pairs that function between 4-10 mM Mg++ as well as another pair, on the same region of the gene that functions best below 4 mM Mg++.

Spend the time "up-front" for the optimisation; it will always be worth the effort and time.

DMSO (10%) reduces the secondary structure of DNA, however, some people show that it can slightly inhibit Taq polymerase - the effect is to reduce the yield of the amplified product (this isn't the case for everyone so other factors must be involved).

Hope this is helpful.


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