low concentration of purified PCR product
Posted 22 March 2012 - 03:45 AM
Posted 22 March 2012 - 03:59 AM
Qiagen makes two kinds of kits for gel extraction, Qiaquick and MinElute, the second allows lower elution volume (10 ul) that can concentrate your sample. But that won't help in your case (even if you use the other), you need 10 times more.
Anyway, for sequencing on ABI machine we need maximum 20 ng of sample (for 1000bp product, less for shorter) in a reaction, so I don't really understand why someone needs 50ng/ul concentration. That would need at lease 500ng band, that's not usual. I would check for other sequencing services that wouldn't need such amounts.
Of course higher the concentration, the better (yours is really low), because you can dilute more and get rid of the contaminants and salts, but 20-30 ng/ul should be enough for everyone as Bill Gates said
I never trust anything that can't be doubted.
Posted 22 March 2012 - 02:48 PM
But, if you only have faint bands, as Trof said you must improve your PCR. You could try more cycles, more optimum annealing temperatures, etc.