Because DNA is soluble in water, is it possible to extract PCR product by dissolving excised gel containing the DNA band of desired length in water, mashing the gel piece with a pestle, centrifuging it and then pipetting out the supernatant ?
$\begingroup$
$\endgroup$
4
asked Aug 13, 2014 at 17:42
-
$\begingroup$ If you mash it with a pestle, you'll likely break the DNA. You can use a low melting point gel and mild heating followed by centrifugation to extract DNA. $\endgroup$– canadianerCommented Aug 13, 2014 at 17:57
-
1$\begingroup$ why not use this? (qiagen.com/products/catalog/sample-technologies/…) or a similar product? I have used it many many times and it can be done very easily! $\endgroup$– Behzad RowshanravanCommented Aug 13, 2014 at 19:08
-
$\begingroup$ @Bez - Well, that would cost around 1-2$ per sample, correct ? $\endgroup$– Anurag MishraCommented Aug 15, 2014 at 14:56
-
$\begingroup$ @AnuragMishra indeed, however, there might be delivery charges but some times institutes bulk buy and sell it in their own stores and their prices are less and sometimes there are institute based purchase pages which sell the products at the discounted price. ps I'm not promoting any brand or products here and I just put it up as a point of reference. $\endgroup$– Behzad RowshanravanCommented Aug 15, 2014 at 16:08
Add a comment
|
1 Answer
$\begingroup$
$\endgroup$
15
Yes, this is possible (and I have done it uncounted number of times) - the method is called "Freeze and squeeze". What you basically do is to run the gel, cut out the band of interest (be careful with the UV light, it causes damage to your DNA and also sunburns, so wear appropriate shielding for your face), dissolve it in a buffer, then freeze it in liquid nitrogen (or the -70°C, doesn't matter) and centrifuge it at room temperature for 10 minutes. During this time the ice melts and the agarose pellets at the bottom. Take the supernatant, do a ethanol precipitation (with glycogen when you only have little DNA) and you have nice and clean DNA. No need for expensive kits and is done approximately in 2 hours.
You can find a very nice and detailed protocol here: "Elution of DNA from Agarose Gels" (The rest of the handbook is also very useful).
-
1$\begingroup$ @Bez: Excitation 290-320 means that it needs to be excited in UV-B (which can cause DNA damage). The only difference is the emission wavelength which is green not orange/red. And that they claim that they are safer than EtBr. $\endgroup$– Chris ♦Commented Aug 13, 2014 at 19:31
-
1$\begingroup$ PA gels are not dissolving when they are subjected to heat. But we rarely use them. $\endgroup$– Chris ♦Commented Aug 14, 2014 at 9:04
-
1
-
1$\begingroup$ @AnuragMishra At maximum speed, 14.000 or whatever your benchtop centrifuge can do. $\endgroup$– Chris ♦Commented Aug 15, 2014 at 21:50
-
1$\begingroup$ @user137 Ihave never compared this systematically with spin columns. And I have mostly used this technique with digested DNA which was subsequently cloned (so mostly digested PCR fragments or pieces of DNA), so I generally had not much material in the beginning. Recovery was ok and cloning with this purified DNA was also not a problem. $\endgroup$– Chris ♦Commented Jun 6, 2018 at 10:39