I already downloaded the fasta format of gene sequences from Oryzabase, how do I make a marker specific only to this particular gene so I can use to identify the presence of this gene from our germplasm collection. Thank you.
The process is quite easy, you need to go to ncbi or directly to primer3, paste your sequence with a code given.
Follow the steps:
1. Put the product size, as needed. Mostly taq will amply upto 1000bp so if u are working with taq pol then ur max product size must be less than 1kb.
2. Primer length, i used max of 25. u need to put max. min and average.
3. GC content as per species, rice will have more than 50%
Click on get primer.
Carefully consider complementarity issues.
Check the attached pic for details....
Jameel is right on. The only thing I would add is to blast the amplicon in NCBI to make sure there are no homologs. Even better, blast the primer sequences and allow a couple mismatches to be certain you are only targeting your GOI.
Anthony how about this?
Primer must have at least 2 total mismatches to unintended targets, including
at least 2 mismatches within the last 5 bps at the 3' end.
Ignore targets that have 6 or more mismatches to the primer.
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