2 replies to this topic

[rephi]

Hi!

I'm working with biotin labeled primers to optain nifH-Gene fragments with PCR for later use as a dot-blot probe. Unfortunately I am getting quite bad results from my PCRs. I was wondering if the biotin Label of my primer maybe changes the annealing temperature. Has anyone experience? I would be glad for hints.

thank you!
rephi

Edited by rephi, 22 March 2010 - 02:00 AM.

[agniesz]

Hi!

I am not an expert, however I do not think it would change the Tm of primers. Biotin  so can disturb during PCR though, so maybe try to change the conditions eg give more primers, try to do gradient PCR to establish the proper Tm

Agnieszka

rephi, on Mar 22 2010, 01:59 AM, said:

Hi!

I'm working with biotin labeled primers to optain nifH-Gene fragments with PCR for later use as a dot-blot probe. Unfortunately I am getting quite bad results from my PCRs. I was wondering if the biotin Label of my primer maybe changes the annealing temperature. Has anyone experience? I would be glad for hints.

thank you!
rephi

[hobglobin]

We used once biotinylated primers...it worked with the standard Tm. Anyway some programs include modifications such as Oligocalc