How to quantitate, to dose first strand cDNA ? - (Jan/11/2006 )

Hello,


I'd like to dose cDNA for real time RT-PCR, I saw 2 different protocols for this ; one describes the formula : D260x33x dilution Factor , and the othe rone uses the formula : D260x50xDilution factor .

What do you think about this and what is the more pertinent ? which formula should I use ?


Thank

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in "Maniatis" they say :

factor 50 for double stranded DNA
factor 40 for single stranded DNA and RNA
around 20 for single stranded oligonucleotides.

I would use 50

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Thanks, But other protocol says 33 (D260x33xDF) ??
Since cDNA is a single strand so, value 40 would be the right one ?!!
Why do you use 50 ?

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.... I wanted to see if you understood
You are right : 40

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So, all your previous measures were false

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I don't care, the most important is that PCR is working

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make sure you clean the cDNA up first or you will get falsely high readings

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Yaaaaaah ! yes right but not always






Do you mean that I should to precipitate my cDNA reaction before to proceed to Real Time PCR ?

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Your cDNA reaction contains a relatively small amount of cDNA (made from the mRNA) along with the mRNA, rRNA, tRNA, and contaminating gDNA. Also dNTPs, RT enzyme, whichever primers you used and salts from the reaction buffer

To accurately quantify the cDNA by OD you must remove all of the above other than the cDNA. In practice this is pretty much impossible - purifying using a spin column or other commercial kit will remove everything but the nucleic acid but as the cDNA will only consist of 1-5% of the total nucleic acid present OD quantification will still be impossible

It is possible to measure cDNA yields using kits like this but it's quite unwieldy and expensive and means your cDNA is labelled

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Hi,

I have a question about that last comment - so you are saying, its important to clean up the cDNA using spin columns or another method.

But its very difficult to quantify cDNA.

Did I get that right?


Which spin column method do you use?

thanks


Do you mean that I should to precipitate my cDNA reaction before to proceed to Real Time PCR ?
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