5 replies to this topic

[graduate student]

Hi,

I am trying to separate 745bp, 646bp, and 655bp fragments (from a PCR reaction) on an agarose gel. I've
separated the 745bp fragment but the other two fragments are not separating on a 2.5%
agarose gel (15cm by 15cm), running at 55V for 3.5 hours. I also want a crisp band because I am
planning to cut these fragments out and extract the DNA for sequencing. I heard running the gel at lower voltage overnight can give you blurry bands. If I run it on a polyacrylamide gel, how would I extract the DNA?   Any suggestions? Please and thank you!

[phage434]

You could try a range of restriction enzymes, some of which may cut one but not the other band.

[graduate student]

Sorry, I forgot to mention that these fragments are splice variants of the gene. There could be other splice variants within this gene that haven't been discovered. So, finding a restriction enzyme to specifically digest one of the band maybe impossible since I don't know the sequence of the splice variant.

[phage434]

I was suggesting random search, not a designed approach.  With the length of your sequence, I'd start with 4 bp cutters.  You could reduce search time by using a mix of enzymes in each trial.

[TJG]

[quote name='graduate student' date='Dec 4 2009, 08:02 PM' post='49663']
Hi,

I think it is a pretty much impossible task to separate 646 and 655 bp fragments by agarose.  You just can’t get fine enough resolution in that size range, regardless of the percentage gel used.  

Running the gel slower/overnight will not improve resolution.  This can give you blurry bands if you run the gel at too low a current.  The pull of the current becomes only marginally stronger than forces of random dissolution and the DNA gets a little bit dissipated giving blurry bands.  Running the gel slower does not improve resolution.

Acrylamide gels are more difficult to work with.  There are protocols and kits to extract DNA from acrylamide but I doubt you’d be able to separate out a 10 bp difference in the 600 bp size range.  Maybe but it might take a lot of trouble and optimizing.

I think your best bet is to cut both bands out together and purify them together.  Now you have  a mix of both bands.  Set up a ligation to clone the PCR products into a plasmid.  Transform bacteria and grow up isolated colonies.  Each colony will now have a different insert.  If the bands you cut out of the gel had roughly equal amounts of DNA then you can expect that roughly half your clones will have originated from each band.  Now you can either miniprep the plasmid DNA from 5 to 10 colonies and cut out the insert and run it on a gel or just go straight to sequencing.  If it’s a 50-50 chance of one insert over the other then you should only need to sequence a few to get to the sequence of both bands.

If you were planning on sequencing the PCR product directly and did not design your primers with restriction sites on the ends or for TA cloning, I’d go back and do that and re-do the PCR with your new primers rather than trying to separate out these bands by acrylamide which would probably end up taking a lot more time and effort.

[martin_poland]

cloning is good idea

if you have different restriction sites inside your inserts you will be able to distinguish them from each other

Edited by martin_poland, 08 December 2009 - 11:51 PM.