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Old 03-19-2014, 03:23 PM   #4
Location: Portugal

Join Date: Apr 2011
Posts: 50

Originally Posted by capsicum View Post
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.
Maybe they use a huge amount of DNA and like that its possible to perform both things at same time. So if you process eveything in the same pcr step, you will get indexed fragments and since you load more its possible to continue to sequencing step.

Originally Posted by capsicum View Post
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.

Originally Posted by capsicum View Post
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).
No, i think you can play around with the starting sample quantity. I just said to load the same amount because its more easy to detect some problems between samples (like contaminants). Do you have enough sample to try to purifiy? Precipitation/purification kits? Your efficiency problem could be correlated with some contaminat in the solution...
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