I am interested in isolating a gene from arabidopsis whose size is 1.2 Kb. I have synthesized cDNA using oligo dT primers, random primers and gene specific primers. But in none of the samples, I am able to amplify my gene of interest.
Did you use the right tissue to do RNA extraction from? Did you check your RNA quantity/ quality?
Did you check if you got sufficient cDNA after synthesis (check on gel or nanodrop)?
After first strand cDNA synthesis, you only get cDNA copies in an equal amount to the RNA that was present. So if your goal is to simply isolate your gene, you have to run a normal PCR for amplification. If your goal is to run qPCR, your shouldn't do this ofcourse (since you alter the quantity of each transcript). You did run a normal PCR for amplification after your cDNA synthesis, did you? If yes, you might try playing around with some of the PCR parameters. Try different annealing temperatures, extension times, denaturing times,...
Also make sure your primers fullfill all of the requirements (GC content, self dimer,...), and that they can actually give your a product. You can for instance check this in Amplifx software. If you can't get primers that meet all requirements (because you can't deviate from your 5' and or 3' ends), try ordering some alternatives (shorter, longer, ...) and test them. If you don't want to waste your cDNA on testing only, you can just get some genomic DNA straight from Arabidopsis seedlings and test if at least your primers anneal. If they are able to amplify the gDNA, they should definitely be able to amplify the shorter cDNA.
By the way, 1.2 Kb isn't so long so unless your primers are really bad, under the right conditions you should be able to get a product.
Dear Sebastjen,
Thank you for your suggestions. I failed in isolating the gene of interest from Arabidopsis cDNA using standard cycling conditions. (94/5 min, 94/30 s, 58/45s, 72/1.2 min, 72/10 min)- 30 cycles.. To my surprise I was able to isolate gene of interest with modified conditions as described in a paper. (95/1.5 min, 94/2 min, 58/30s, 72/2.3 min, 72/10 min) - 40 cycles. The change was in denaturation and extension time. What could be the possible reason for this?
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