problems regarding amplifying a 1.7 kb mRNA seq - (Mar/17/2014 )
i want to clone 1.7 kb mRNA seq. here i followed two set of primers (from published papers). i was tried to convert RNA from cDNA by using random hexamers. i didn't get amplification of primers from my gene of interest. i dont know the problem exactly with cDNA quality or primers and also i used internal control GAPDH for to check the cDNA. i got amplication in internal control. i was tried different gradient, different concentration of cDNA and also i used pcr additives. plz find a problem and give me a idea to do amplify my gene.here i attached some details about pcr reactions ,
maser mix (emerald, green Takara)- 10
F primer (10pm) - 1µL
R. primer (10pm)- 1µL
Template (cDNA )- 2µL
AMQ - 5µL
PCR conditions :
Initial denaturation - 94˚c- 5mins
denaturation - 94˚c-1 min
extension72˚c- 2 min
final extension 74˚c- 15 mins
34 cycles, hold 4˚c- forever
As random hexamers bind to the RNA randomly, this can interfere with the production of full length cDNA, so you would be better off using either gene specific primers or oligodT for your cDNA production.
i was tried by using gene specific primer. but i didn't get amplification. i never used oligodT for to synthesis and i had a doubt whether a problem with gene specific primers. how to check whether primers are good?
You can compare the primers to the sequence for your gene found on Genbank.