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PCR product loss on cleanup

PCR clean up

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#1 Questionner

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Posted 20 April 2012 - 11:12 AM

I did two PCR reactions yielding products of about 1.5kb. Both looked to be the same concentration on a gel and beautifully clean. I then used the Clean and Concentrator kit from Zymoresearch eluting in 12ul total. On measuring the concentration, one was at 200ng/ul, the other was at 12ng/ul. This is the second time I've experienced yield loss with this PCR reaction (specifically this product). Any one know why I could lose DNA for one product and not the other? Anyone experienced something similar and what did they do about it? I am going to use restriction enzymes on both products and then need to clean them again for ligation so I can't afford this type of loss even if I can digest in the PCR buffer. Any suggestions welcomed.

#2 phage434

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Posted 20 April 2012 - 11:28 AM

Make very sure you are spinning the columns dry, after dumping the wash from the collection tube. Small amounts of wash will inhibit the elution of the product from the column. This is especially an issue with small elution volumes. You could elute a second time, which would work even if the first elution merely diluted the ethanol left in the column. You need to clean up after PCR and before digestion, but you do not need to clean up after digestion if you can heat kill your restriction enzymes. Choose enzymes that can be heat killed.

#3 Questionner

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Posted 21 April 2012 - 12:26 PM

Thanks phage434, I will try an extra spin to dry the column next time.




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