I have PCR-ed some DNA samples which I need to perform gel extraction on. These DNA samples will subsequently be used for restriction digestion and ligation over the next week. I was advised to elute my DNA in water to increase the yield but I'm not sure if that is a good idea. Is it better to elute using the gel extraction kit elution buffer?
The reason you were told to use water instead of elution buffer is that sometimes, the elution buffer has EDTA or some other substance like that. If you plan on performing ligation on such purified fragments, then you should use water instead of elution buffer because EDTA inhibits the reaction. If you are worried about the yield:
- Let it penetrate the membrane for 5 minutes instead of 1 minute as the protocol usually says
- Do so at 37ºC instead of at room temperature
- Do this twice, meaning: collect the DNA after the first centrifugation and place it back in the membrane for 5 minutes at 37ºC again.
All of this helps increase the yield. We use water instead of elution buffer all the time in our lab (because we want to ligate them to plasmids afterwards) and we are fine.
Cheers.
Maziar S. Ardejani ah okay. You have a point. Thank you for your input!
The reason you were told to use water instead of elution buffer is that sometimes, the elution buffer has EDTA or some other substance like that. If you plan on performing ligation on such purified fragments, then you should use water instead of elution buffer because EDTA inhibits the reaction. If you are worried about the yield:
- Let it penetrate the membrane for 5 minutes instead of 1 minute as the protocol usually says
- Do so at 37ºC instead of at room temperature
- Do this twice, meaning: collect the DNA after the first centrifugation and place it back in the membrane for 5 minutes at 37ºC again.
All of this helps increase the yield. We use water instead of elution buffer all the time in our lab (because we want to ligate them to plasmids afterwards) and we are fine.
Cheers.
If you are planning to carry out restriction digest of eluted DNA, it is not a good idea to use water: In pure water, DNA is singlestranded. This was published ages ago, but people seem to have forgotten.
Geoff Margison, would you mind putting up a paper reference. There aren't many publications regarding DNA interactions in pure water
You have to go back a long way! One by Phil Lawley (BBA, 21 (1956) 481-488) I have a PDF of. A summary/memoire by Rene Thomas ( Gene, 135 (1993) 77-79) contains other relevant refs. Note that after elution with pure water, partial renaturation will occrr when sodium ions are added. But my advice is to elute with the kit elution buffer, or TE, which is what that might well be.
The eluted DNA will be more stable in a buffered solution , I only used Water for eluting when send for sequencing
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