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Very low yields for >10kb fragment gel extractions

dna purification gel extraction cloning

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#1 naglemi

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Posted 18 February 2017 - 12:04 AM

I've mostly been using Qiagen Qiaex II kits and have been getting very low yields for extraction of large fragments. My yields for small fragments are decent. I've used fresh solutions and have made sure lids were kept tight and the right amount of ethanol was added to P1.

I add two volumes water for fragments >4kb during the gel dissolution step, as instructed. Otherwise, there's no difference between how I extract large and small fragments.

I've also used Zymogen Gel Extraction kits and have gotten lots of guanidine. I've seen some posts saying that will happen with these columns and it doesn't interfere with downstream cloning, but I think it might be interfering with a Gibson Assembly.

At this point, I'm stumped. I have seen some posts saying Qiaex II kits won't give a good yield for >10kb fragments... What should I do then? I need to get a high yield for 15kb gel extractions and would greatly appreciate any advice. I can get other kits if needed, or prepare the solutions myself.

Thanks for the tips.



#2 bob1

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Posted 21 February 2017 - 08:29 AM

Gel extraction yields are usually low relative to the input, so this probably isn't your fault. The reason big fragments don't isolate well is that they are so big that they get physically stuck in the matrix used for the columns, so no amount of elution will get them out. You could try other methods of gel extraction such as just excising the band, freezing the gel then spinning down to extract the DNA containing supernate.

 

You may also want to consider running the band into a hole cut in the gel, then sucking out the fluid containing it and precipitating from there. Check out the protocol in Sambrook's "Molecular Cloning".







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