seperating bands for sequencing

[kermitdfrog]

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Does any one have any suggestions for separating bands on a gel so that they can be cut out, purified and sequenced. The difference in size is about 20-50 bp, with the lower band being around 210 bp (already sequenced). I've tried several 2-3% agarose gels, but I keep getting both bands appearing when I cut out the top band (the one I want to sequence) purify the gel slice and PCR the purified product. I'm assuming that smearing is contaminating the gel slice. I've also tried 7% acrylamide, but the bands are way to close to even attempt cutting the top one out.

 

[Mitro]

Clone the two fragments into a plasmid and sequence off it. It takes a little more time - but you'll be 100% sure that you're only sequencing one product.

 

[mendel121]

Clone the two fragments into a plasmid and sequence off it. It takes a little more time - but you'll be 100% sure that you're only sequencing one product.

I think mitro is right, 20-50 bp is too close for gel separation, cloning is a good suggestion pGEM T-Easy from Promega is a good solution if your looking for a cloning system

 

[kermitdfrog]

Thanks for responding. I should have mentioned I already tried coloning, and some of the clones produced either the lower more dominate band or both. When I tried to get a sequence from the ones with double bands they pretty much gave me junk. It is definetely something that I'm going to have to revist, although I guess I was keeping my fingers crossed for some easy gel extraction method. My PI really wants me to follow this, although I feel like I'm getting no where and putting in way too many hours.

 

[Mitro]

Your clones shouldn't have multiple bands - that's kinda weird. You might want to try one of invitrogens super lazy topo cloning kits - they are a godsend. I've never had the problem you're describing using one of their kits, even when cloning multiple products (10+). If you provide more details, it might be easier to troubleshoot.

Alternatively, you can always design new primers.

Good luck.

 

[Gut Shot]

Does any one have any suggestions for separating bands on a gel so that they can be cut out, purified and sequenced. The difference in size is about 20-50 bp, with the lower band being around 210 bp (already sequenced). I've tried several 2-3% agarose gels, but I keep getting both bands appearing when I cut out the top band (the one I want to sequence) purify the gel slice and PCR the purified product. I'm assuming that smearing is contaminating the gel slice. I've also tried 7% acrylamide, but the bands are way to close to even attempt cutting the top one out.

You could toss some p33 radiolabeled dATP into the PCR mix and then separate the bands on a sequencing gel (a la that wonderful technique, differential display). Having to do that would suck, but you could get 1 bp resolution on the separation.

Then you just dry the gel on some paper, cut out the band of interest, elute into some water and repeat the PCR.

 

[Gut Shot]

I should have mentioned I already tried coloning, and some of the clones produced either the lower more dominate band or both.

That's bizarre. Are you sure that the upper band isn't just artifact? You could try a 0.5-0.8% gel, run very slowly.

 

[kermitdfrog]

It may well be an artifact (something I'm really starting to think), but there are lots of signs pointing to the fact that it may also be real.
Some details:
1)I tried using a DNase protocol with my RNA before making my cDNA and I still got the extra band, so I don't think it's genomic DNA contamination.
2)I also tried a new set of primers and got the same multi band pattern.
3)When I get multiple bands there are actually three bands. The two I've mentioned and a lower one that is about 140 bp. I'm just ignoring this lower band, because it is very easy to separate. It is the middle band being too close to the upper band that seems to be my problem.
4)It did seem strange to me that three of my clones (using PGEM-T easy vector system protocol; 13 colonies, and 8 that created minipreps) gave all three bands, while the rest produced only the middle and top band. The top band was extracted for cloning, which at the time was the best separation that I could get, however there was some obvious streaking. The thinking behind extracting that band was hoping that one of the clones would only have the top band. The lower two bands have already been sequenced. Since that time by playing around with the annealing temp I've been able to get much better separation, although I'm still having the streaking issue. It is apparent because when ever I extract the top band, purify it and run a PCR I get the multiple bands appearing, which isn't the case now when I extract either the middle or lower bands from an acrylamide gel as I only get those bands appearing. (I haven't been able to extract only the top band from an acrylamide gel yet).
5)I've used RNA extracted from four different human tissues, the top band consistently appears in two of them and faintly in the other two.
6)In a paper describing the middle band as a new splice form, with the lower band already previously described, a figure clearly shows the third top band in several different human tissues, however it is very faint and not mentioned at all. The paper was published several years ago, with nothing being published since then about a third splice variant.

I like the idea of trying a lower % gel, I may try running a 5 or 6% acrylamide gel, and I also may try the .5-.8 agarose (running very slowly, although with the size I'm afraid my product may just run off). I'll probably also retry the cloning once I can extract a top band that I feel fairly confident has avoided the streaking problem or get good enough separation with the acrylamide. Another possibility that I was thinking of is that there could be some sort of polymorphism where some DNA is folding, making it more difficult for it to migrate through the gel, which makes it appear to be a third band. However the consistency with which I'm getting this top band, and the fact that I got it in different tissues, my clones, and others have seen it makes me question that theory a little. I really appreciated everyone's help and suggestions.

 

[vcatz]

Have you tried metaphor agarose? It is different than regular agarose - it is used for fine resoultion of DNA bands. It think you can separate bands with a size difference of ~20 to 50 nt using metaphor agarose. We use it for microsatelite genotyping and can resolve such differences.

 

[kermitdfrog]

That's definetely something I'll consider. I know a few people who think the world of cambrex agarose products.

Have you tried metaphor agarose? It is different than regular agarose - it is used for fine resoultion of DNA bands. It think you can separate bands with a size difference of ~20 to 50 nt using metaphor agarose. We use it for microsatelite genotyping and can resolve such differences.

 

[Ursus Martimus]

Acrylaminde gels used to be used all the time. RNA would be separated out and the transcripts would be isolated from the gel and injected for expression in oocytes. The yield is not as good as from agarose and the protocols are a bit more cumbersome. YOu should be able to separate seuences within 2-3 bp's with the right concentration gel, say 7-8%. That is how they used to gel purify oligos for PCR primers. Sometime old school still rocks.