Should I always dilute cDNA before PCR amplification?

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Britta Boserup Thestrup

Hi Renas Isa

I would reconmend to dilute the cDNA samles before PCR. High DNA concentration could prevent succesfull amplification.

Best regards Britta Boserup Thestrup

Katie A Burnette

You might need do, but that depends on the concentration of your cDNA and the abundance of your target gene. Biorad has good information on qPCR experimental design.

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5859.pdf

Renas Isa

Britta Boserup Thestrup Katie A Burnette Md Abdur Rahim thanks alot for the answers. As I know, NanoDrop cant measure the cDNA concentration. Is there any method to know the quality and quantity of cDNA before performing PCR (conventional)?

A Qbit machine will give you a more precise measurement of the quantity of your cDNA than a nano drop. The A260/280 ratio will tell you a bit about the quality, but the only way to know for sure is to use the cDNA in a reaction.