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Old 03-14-2014, 01:32 AM   #2
DRYTCYV
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Location: Portugal

Join Date: Apr 2011
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Quote:
Originally Posted by capsicum View Post
1. Does anyone carry out the first and second PCR steps together on the one reaction? I read about someone doing this, but I can't seem to find it again.
- 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


Quote:
Originally Posted by capsicum View Post
2. What QC do you do on your primary and secondary PCRs? How do you deem a success or failure? Just band visibility on a gel?
- Qubit and gel (1st),
- Qubit, gel and qPCR (2nd) to normalize.

Quote:
Originally Posted by capsicum View Post
3. How do you deal with a project where some samples show very strong amplification and others showed only just sufficient amplification for sequencing? Do you optimise the PCR (by diluting the template, for example) to increase amplification of the poorly performing samples, or just sequence them anyway if there's enough product to do so?
- 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)

Quote:
Originally Posted by capsicum View Post
4. If you dilute your samples to obtain better amplification, do you think this needs to be done for all samples that will be compared? I.e. do you think it will bias the results if you sequence samples that have been treated differently in this way and then compare them? If not, then do you think it's legitimate to compare samples that have radically different amplification efficiencies?


5. Do you think it's important to normalise the input DNA concentrations for samples that will be compared? What about the input source material? We have some projects where samples that need to be compared might contain DNA concentrations that differ by 100x or more.
- 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 hope these will help you
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