do you purify fragments from "regular" agarose gels ?

colossus... s535290 at
Wed Oct 1 23:50:16 EST 1997

Hi all,

I am encountering a little bit of a problem in trying to clone a
particularly pesky DNA fragment. My custom-made vector contains the
luciferase coding region as an NcoI/HindIII fragment. What I am trying to
do right now, is to excise luciferase, and replace it with my gene of
interest. I can excise luciferase from the vector, as seen on a gel. 

1) it seems that the vector is what it should be : only the doubly cut
vector releases the luciferase coding sequence. 

2) When I try to ligate the doubly cut vector to either the excised
luciferase coding sequence, or to the fragment that I am trying to clone
(also an NcoI/HindIII fragment), I get nothing. 

3) The intact vector transforms just fine.

4) the ligase buffer and ligase itself have worked for other endeavours
lately, so I have no reason to doubt them.

I use standard sticky-end ligation conditions. I haven't quantified the
vector or insert DNAs accurately, should I be worrying about vector/insert
ratios ?

I have tried to gel purify my fragments in a couple of different ways
because some folks have complained in the past that different gel
purification systems can yield DNA that will not ligate. But I have no
reason to believe this is the culprit. We *have* recently changed agarose
suppliers...and I am just wondering if this could be a problem. Some
people always swear by using the more purified agarose derivatives when
gel purifying stuff, but I have never had trouble in the past.

any ideas ?



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