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MatK gene region in angiosperm

Dear all,

I am currently working on DNA Barcoding of some plants (belonging to Angiosperm). Fistly ı did not use DMSO for MatK gene region. But sequence results are not good and then ı use the DMSO, but so now non-spesific band is occurring.  Does anyone know why this might be? How can I do ??

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Replies to This Discussion

Permalink Reply by Rasika Bhagwat on May 28, 2012 at 10:28am

Hi

Which primers you are using, becoz I am also facing the same problem.

Rasika

Permalink Reply by Handan on May 29, 2012 at 2:31am

Hi,

I use primers trnK-2R and trnK3914F ... ;Is it same your primers??

Permalink Reply by Paula on May 29, 2012 at 2:21am

I read in an article that DMSO lowers the annealing temperature of your primers, so if you add DMSO, you will have to adjust your cycling conditions.

I use primers 1248R (Tm 52.5C) and 472F (Tm 57.8C) and my PCR annealing temp is 53.7 when I add DMSO, and I have excellent amplification. Good luck! 

Permalink Reply by Handan on May 29, 2012 at 2:36am

I understood, I will try to change my temperature. I hope,  I get a good amplification...


thank you so much ..

Permalink Reply by Paula on May 29, 2012 at 2:47am

Good luck, I hope the advice helped!

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