SOS: Gel purification of small size DNA - (Sep/29/2005 )

hi,guys
i have purified 2 bands with OMEGA kit,but can't see anything by gel detection,i don't know what was going wrong.Is it difficult to purify small size DNA with kit ?can i use phenol/chloroform extraction and ethanol prcipitation but not using PCR purification kit ? i need your hope,thanks in advance!

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What is the size of those fragments?

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Some really small DNAs can be difficult to gel purify, so it is important that we know the size. You are going to have a real difficult time seeing small DNAs on a gel with Ethidium staining. That is normal given the small molar amount of DNA you will have run.

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thank you ,two guys
the size is 125bp and 168bp,respectively.

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To visualize on a gel use a 2% agarose gel made in 0.5xTBE and it will be perfectly fine

For the extraction Quiagen or Macherey Nagel kits can do it nicely I'm routinely purify this type of pCR products !

Pesji

The picture is such agarose gel made with cheap equipment in Ghana (during my pCR study overthere)

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Yup, that would work for PCR products, but you have to run out quite a high concentration to detect it. If you increase the amount you purify, you should be able to see it on such a gel. It should also be no problem to purify with standard gel purification techniques. Just take it easy on the ethidium there bud!

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My recommendation is to use 3% low melt agarose and freeze squeeze purify the DNA. Freeze squeeze works very efficiently on small fragments and it is hard to be the price

Daniel

Automated DNA sequencing troubleshooting

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