4 replies to this topic

[jonny]

Hi,

I am screening for microsatellites and am using size-selected genomic DNA (between 300 and 900bp) for ligation into a 3000bp vector.  The genomic DNA was digested, run on agarose, cut, and gel-purified.  I have no problem with the transformation.  However, mostly only small pieces of about 150-250 successfully ligated.  Larger pieces of my target size (500-600 bp) are not ligating as frequently.  Even when I cut further up on the gel (between 800 and 1400 bp) my average insert size is still only between 150 and 200 bp.  Does anyone have any advice for this ligation bias toward smaller fragments?

[peixumol]

alter your molar ratio of large to small insers ,in other words ,increase the relative amout of  your larger inserts . you konw ,the mole number of large DNA fragment is much smaller than small one if they are in the same microgram .in addtion,the ligation effiency is naturally lower for large ones. wish your  following success!

[jonny]

I don't know of an easy way to do this since all of the fragments are pooled together after gel-purification....and there are of course more smaller fragments in the mix.  I certainly want to increase the ratio of larger fragments, but I'm not sure how.....

[peixumol]

you don't know?it is extremely easy!load enough amount of digested DNA,then cut and recycle the larger and smaller ones seperately.mix the two with a larger  amount of the former...just as simple

[jonny]

peixumol, on Jan 14 2005, 02:38 AM, said:

you don't know?it is extremely easy!load enough amount of digested DNA,then cut and recycle the larger and smaller ones seperately.mix the two with a larger  amount of the former...just as simple

Thank you, but  I think maybe you misunderstood my question.  I have already size-selected my DNA.  The digest is run on an agarose gel, the size-range that I am interested in (300-900bp) is size-selected by cutting the gel, and this gel piece is gel purified.  However, my problem is that even though I cut between 300bp and 900bp or even between 800bp and 1400bp, there are STILL smaller fragments of 150bp which must be migrating in this range and these are the pieces that are ligating instead of the larger ones between 300 and 900.....anyway, thanks for trying!