can you ligate gel slices directly? - (Jan/25/2006 )

i have tried to purify DNA from the gel by using phenol/chloroform extraction, somehow methods says that i should add 5 volumes of buffer to it to melt it....and since gel slice that i have got was around 1 ml i ended up extracting 6ml of DNA... ....yes i know im crazy but what could I do?! next time ill try freeze and crush method.....but someone on the web posted a method of ligating DNA directly from the gel...just melt it and take like 10ul of it and ligate....what do you think?
sorry it says like take 10ul of PCR insert digested gel slice and 5ul of vector digested gel slice and ligate....

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How could you possible end up with 1 ml gel slice?
How much insert have you calculated that you need in your cloning?

To ligate directly from the gel, you need to know both.
One of the reasons people extract DNA from gels is to be able to increase the concentration.
The other is that they suspect there might be compounds in the gel, or buffer, that inhibits the ligation reaction.

The important question is: How much DNA is there in your gel?
A normal band in a gel would maybe be around 1-2 mm x 4-5 mm, and the tickness of the gel - maybe 5-6 mm. This would give around 50-60 microlitres of gelslice, and if the band is really intense there might be up to 200 ng DNA here. 10 µl would in this best case senario give 30-40 ng DNA to clone.
More than enough. But then you need to cut the band very carefully, to avoid extra gel, because all extra gel dilutes the DNA.

If the band is not that intence, - maybe you can hardly see it in your gel. Then the amount of DNA is more likely to approach 20 ng, and 10 out of 50 µl is suddenly only 4 ng.

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perhaps you could invest in a Wizard SV gel purification kit from Promega. It may make you purifications much less work. Alternatively a cheap method or isolating your dna from the gel is to cut out your gel peice (as small as possible while still containing the whole band) and then:
1) cut a filtered pipette tip so it will fit just right inside a 1.5 ml microfuge tube
2) put about 20-50uL of sterile water in the top of the tip (which is in the tube) and spin it down for ~1 minute
3) remove the water from the tube and anything remaining in the bottom of the pipette tip
4) place your gel peice in the top of the tip (so it is sittin on top of the filter)
5) spin the tip in the tube again and collect the liquid. The liquid contains your DNA. The solid part of the gel will remain on top of the filter.

If desired ethanol precipitate to remove EtBr or go directly to ligation.

This procedure usually takes about 3-4 minutes and is cheap. Best part is you don't have to do calculations.

-Cap

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thanx a lot..its my first time cutting from the gel so obviuosly i have cut more than enough....but i have loaded 200ul of PCR mix from the beginning....i suspected it would contain around 200ng of DNA.....maybe loading just 50ug (one PCR) would be enough? thanx about the pipette tip....but what kind of filter do I use? is that what you mean by filtered pipet tip? seems really easy want to try that one....

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You just use a regular sterile filtered pipette tip. They look like regular p100 tips but with a filter to prevent contamination of your pipette. Here is a link to the picture of one sold by Fisher. Odds are you have them in your lab. I believe we used p100s.

200 uL of PCR mix is probably too large of an amount to try to put all in one well. I generally don't go over 25uL (I usually do only 25uL PCR reactions to save reagents). Any other questions feel free to post.

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Captain DNA, thanx a lot...but i have done transformation and i ve got colonies... so im checking them now by PCR....but negative control (no plasmid) has one colony too ... ..so i suspect there is some kind of contamination still anyway i guess not all of my sample colonies are contamination of course....so i hope ill have some luck and get my insert....about the filtered tips...we dont have them here in the lab... so if my ligation didnt work this time ill try to persue my prof to order them..

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This is an easy and inexpensive method but I still personally use the Promega Wizard SV purification for most of my extractions. With the pipette tip method don't forget to ethanol precipitate afterwards to concentrate your DNA and remove EtBr if needed.

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Ugh, I used to have a protocol for directly ligating two gel slices, but I can't find it anywhere!

The protocol came from a lab at Harvard, so maybe some combination of 'harvard.edu cloning ligation gel slices' would work (no quotes).

-Hank

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I have cut gel slices, run them through glass wool, then ligated the juice together and gotten clones...if it is straightforward and should be pretty easy. if the clone is tricky at all or really important and time-critical, I do it with a little more stringent purification

just make sure not more than 1/2 of your overall ligation mix comes from the gel slice, or the ligation is inhibited

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