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Old 03-16-2014, 02:23 PM   #3
capsicum
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Quote:
Originally Posted by DRYTCYV View Post
- The 2nd PCR reaction efficiency is influenced from the 1st reaction,since the insertion of adaptors depend of a specific sequence from the 1st step
Yes, but how reliable do you normally find them? Some people must find them reliable enough to perform both at the same time (in the same reaction). I'd be worried that the product from the 1st PCR was too little. I guess you'd see that reflected in the final product (or lack of it) anyway.

Quote:
Originally Posted by DRYTCYV View Post
- Qubit and gel (1st),
- Qubit, gel and qPCR (2nd) to normalize.
This is what we'd planned to do too, except for the gel... many of our samples are far to low in concentration to visualise on a gel.


Quote:
Originally Posted by DRYTCYV View Post
- Be sure that the sample quality is the same, run a gel (1%) before you start and check the integrity. And of course load the same dna amount (measured by Qubit)
We can't always be sure of the quality because the DNA comes from many different sources (but all samples to be compared to each other) and usually the DNA is very low concentration (just a few ng/uL)

Quote:
Originally Posted by DRYTCYV View Post
- The protocol information for the 1st pcr its 2,5 ul of a 5ng/ul sample. So if you want to compare samples the best thing its to proceed the same way. Load the same, in the begining, ->clean-up->gel -> 2nd PCR ->clean-up Qubit+gel+qpcr and normalize
I know Illumina has suggested that mass and volume, but does it really make a difference? Theoretically, do you think it is more scientifically sound to use the same DNA mass or the same total original sample mass/volume before DNA extraction? I don't think it matters for the purposes of comparison... perhaps only for technical reasons in order to get the PCR to amplify efficiently. Often we have to dilute our samples before they can be amplified, even when the DNA concentration is low (lots of inhibitors that are very hard to remove, and very low cell content to begin with).
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