I am designing primer for xylose reductase and xylitol dehydrogenase gene. Can anyone suggest me what parameters should be their in primer designing.
If anybody having protocol please provide me.
Generally, We keep parameters like this
(1) GC: 40 to 55(max)- dont go beyond
(2) Tm: 50 to 60
(3) Length: It depends on gene length by in safe side, It should be around 20 to 24.
find the attachment for the clear primer designing protocol.
Remember that you are dealing with TWO primers in PCR. Their Tm’s should be within
5°C of each other; the closer the better ....
Hi, If I need primers I usually use : primer 3: http://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm
Primers TM around 50 - 60 for both
and 55% CG
Céline
You can use from IDT, Primer3plus, Oligocalc, reverse complement websites for primer designing. you must mark this parameters:
GC content: 40-55 %
Length: 20-25 bp
Tm: 50-60 (both primer have a same Tm)
At least one C or G at the 3' end.
What are you intending to use the primers for? Primers for QPCR have different design considerations than primers for genotyping or primers for cloning full-length genes. One parameter common to all of those applications is to check your primers for specificity (BLAST them against your target organism to determine if they are gene-specific).
Make sure your primer (forward or reverse) won't anneal to 2 or more places (such as "repeats") in your gene (if your gene has repeats). Make sure they are unique.
Hi Amy Klocko,
I am designing primer to just isolate full length gene for cloning purpose.
Well, if you are isolating the full length of your gene, there are not too much to choose.. You need to have one primer to cover the beginning of the gene and the other primer to cover the end of the gene. In this case, more important parameters will be-- as people suggested above, (1) similar Tm for both primers, (2) set Tm around 50-60C. Thanks to Amy's question.
@ Mahboubeh,
Maybe I am not aware of it, but why we need at least one C or G at the 3' end of the primers? Just curious. Thanks.
I mean that at least one G or C there must have been at 3' end to firm and strong binding because of triple hydrogen bonds between G and C than A and T. Elongation of new strand continue from 3' end so G or C existing make surely binding and extension.
If you pay attention we select 1 for GC clamp parameter in primer designing websites such as IDT for same reason.
Hi Nilesh Kumar Sharma,
Since you are designing primers for producing full-length clones the primer locations are set. You can vary the lengths to try and meet the parameters as suggested by others (annealing temp, GC content etc.). There are lots of programs available for checking these parameters, I normally use the OligoAnalyzer tool on the IDT website (it’s where I purchase primers). www.idtdna.com
It’s user-friendly and automatically provides the reverse complement to any sequence you input.
Amy
@ Mahboubeh,
Thanks for replying and mentioned the 'GC clamp' concept.
Frank
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