I tried to identify 4039bp mRNA sequence by dividing the whole sequence into 6 fragments and designing primers for each of them. But two fragments in this sequence couldn't amplified by PCR reaction. I re-designed the primers for unknown part based on received sequences. But I couldn't amplify it. I tried to optimize the primers based on primer concentration , cDNA concentration, annealing temperature ..etc. I used the CDS sequence of amino acid sequence in NCBI as reference sequence for primer designing. Please can anyone suggest how to solve my problem.
You may be working off an incorrect sequence. If you sequence the amplimers that do work you will get the correct sequence and you can design primers from within your known sequence across the problem areas
Try sequencing from both directions using regions of your sequence to design primers. You're doing an "old-school primer walk" across your sequence.
As Paul Rutland said, use YOUR sequence to design your primers, the NCBI reference sequence may not be a good match.
I guess your mRNA had many isomers or variants and might you have designed the primers in a region which is not 100% identical, I suggest to align the possible isomers from your target available in NCBI and design a new primer set (to amplify the targeted region) in the conserved regions, and if possible you can perform an additional nested PCR step using the primer set to amplify a large piece of DNA followed by PCR product purification step (possibly use EXOSAP IT kit), then reuse the amplified PCR product as a target in your experiment with the new Primers.
I support the suggestion of Katie A Burnette to sequence your targets in both directions, using a single primer (Forward or reverse) in a separate well.
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