Hi, I want to know how to design primer pair for A, B and D genome of wheat together, in order to check expression level in different organ?
Here is some good starter tips
https://www.thermofisher.com/tr/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html
Here is a good web-based software to help you design them
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Alternatively, you can use this software to design primers (free)
http://perlprimer.sourceforge.net/
I've had good experiences with both.
When you are done make sure you do a BLAST search to double check your primers are specific to your sequences
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Hi I need to design a pair of primer for wheat A, B & D genome only at conserved region, but when i use primer 5 to design primers, it give me best result a show in attachment that my forward primer contain one divergence but i need to make my primers only at conserved sequences. My question is this is it cause any problem? and please check Tm and GC content % , is it ok?
Dear friend check the attached link
If you have problem contact me
http://www.sigmaaldrich.com/life-science/custom-oligos.html
GC content is ok (between 40 and 60 %), Tm seems ok (between 52 - 58 °C, difference below 5 °C). End your primers (3') with a G or C (no more than 3 G/C in the 5 last bases). You said you have one mismatch (is that what you mean?). In this case, prefeer mismatching A-C or G-T.
@Mathieu Dondelinger no, i actually make primers for only conserved regions not divergent region, but on conserved region i have only one divergence, it it effect amplification? and what kind of annealing temp i use for this pair of primer?
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