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primers with tags how to decide wich nucleotides


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#1 gala84

gala84

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Posted 12 August 2011 - 03:55 AM

Hi to everybody, I'm new in this forum and I'm also new in molecular biology.
I work on marine bacteria involved in Nitrogen Cycle and from environmental samples I have to do PCR on specific functional genes. I have to work both with DNA than cDNA and on these selected amplicons do pyrosequencing 454. To distinguish the samples from DNA and cDNA I should add a tag of 5 nucleotids for each primer (forward and reverse) that needs to be different for DNA and cDNA. So when I'll have my sequences I know from which samples they come.
Probably i didn't explain it very well.... sorry!!!!

My questions are:

1. With these tags, since the lenght is higher, is it necessary to modified the annealing temeperature???
2. The tags mustn't do dimers and secondary structures with the sequence. How I have to choose the correct nucleotides to put at the end of each primers? The company where I order them can do this? Or I have to do by myself? and How?

thank you in advance

Gala




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