Most of the primers we have designed and purchased have shown negative gene expression on BM samples- although stated otherwise in the literature.
So i'm wondering what could be the problem ?
Thanks !
Hello Dr. Bodoor
i suggest you read these
1- An Introduction to PCR Primer Design and Optimization of Amplification Reactions
Article An Introduction to PCR Primer Design and Optimization of Amp...
2- General Concepts for PCR Primer Design
https://genome.cshlp.org/content/3/3/S30.full.pdf
3- PCR Assay Design and Optimization
http://www.bio-rad.com/zh-cn/applications-technologies/pcr-assay-design-optimization?ID=LUSO1VKG4
Watson, Gilman, et al's Recombinant DNA 2nd Edition was the ultimate source for me on all things DNA.
As for polymerases and protocols, Roche put out a trouble shooting flip-book (like 4in by 6in) guide 20-odd years ago that came in handy with difficult PCRs, but has since been replaced with everything being online these days.
- If certain primers work on samples and some do not, that at least rules out sample related issues (i.e. contaminants in samples inhibiting PCR) and points to primer vagaries
- There are a few simple rules you need to obey when designing primers but these are more guides than gospel to follow verbatim
- Accordingly, find attached my SOP I devised to train students explaining such rules and freeware to use in detail
- In addition, see body of this answer for prior answers I have put together on this subject
- https://www.researchgate.net/post/Which_are_the_key_points_to_during_design_a_primer#view=5672d97d7dfbf9da3a8b4567
- (Top answer by me)
Laurence Stuart Dawkins-Hall
I would recommend your guidelines for this alone "Avoid 3' Terminal T."
Been telling people that for years.
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