Hello,
I'm trying to design RT primer for dumbbell-PCR RT-PCR step. As I understand primer should begin from the place where adapter stops, but I don't understand, what is the sense of additional nucleotids.
Could someone explain me how I should design my RT primer for dumbbell-PCR (5'-Db-PCR). Should I do it manually or is it a special tool for this?
I tend to use Primer3 too, but when doing site-directed mutagenesis, I use the online Agilent Stratagene QuikChange Primer Design Program, since I use their site-directed mutagenesis kit and they recommend it - it's worked fine for me for that purpose so far: http://www.genomics.agilent.com
Hello Darja,
We use Snapgene ( 1 month free version available), or use serial clonar (free).
Additional nucleotides are important because DNA sequencing do not read initial nucleotides properly.
Add adaptors, design primers for adaptors.
Good luck...
I tend to use Primer3 too, but when doing site-directed mutagenesis, I use the online Agilent Stratagene QuikChange Primer Design Program, since I use their site-directed mutagenesis kit and they recommend it - it's worked fine for me for that purpose so far: http://www.genomics.agilent.com
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