When I ran my PCR products on 1% of agarose gel, I got quite unclear and thin bands, but I still managed to cut them out for purification. then I purified them using qiagen purification kit. Then I quantified them using nanodrop spectrophotometer and I got the reading around 5-9 ng/uL. I also check them on gel electrophoresis (loading dye 1 uL and DNA 5 uL) and only 2 bands appeared out of 10 samples with very small and tiny bands. I cannot sent my samples for seqeuncing yet because they require at least 50 ng/uL of purified PCR product. Can someone help me how to improve my DNA concentrations and have clear and bright bands?
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#2
Trof
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Posted 22 March 2012 - 03:59 AM
Generaly, improve your PCR to get brighter bands. You can also make more reactions and join the gel slices if they don't weight over the limit.
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
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
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#3
HOYAJM
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Posted 22 March 2012 - 02:48 PM
Instead of gel purification, which I have found to not be the most efficient in yield, you could try a PCR clean-up column. Zymo makes a DNA clean-up and concentrater spin column shown here
http://www.zymoresea...-concentrator-5
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.
http://www.zymoresea...-concentrator-5
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.
