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generating cDNA from human brain RNA for cloning
I have extracted some RNA from human brain samples and want to generate cDNA from this. I prepared cDNA using random hexamers, oligo(dT) and gene specific primers. Next I try to amplify the genes of my interest, but do not get any product after PCR. All i see is the low molecualr weight primers or primer dimers.
For gene specific primers I used 2 ul of 1uM antisence primer in a 20 ul reaction volume and i do RT for 50 min at 55 C.
The genes of my interest range in size from about 1Kb to about 6 Kb.
Any suggestions as to what I am doing wrong or what else I can try.
Re: generating cDNA from human brain RNA for cloning
Did you heat you RNA template before RT? That's done in order to get rid of RNA's secondary structure (if there's any).
You can also run your RNA in a gel to make sure you do have RNA, or use an spectophotometer. I don't have much experience in this regarding RNA but DNA is read at 260 nm.
I hope this help you
|brain , cdna , cloning , generating , human , rna|
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