When Low Melt bands are contaminated???

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Jan 5 13:55:05 EST 1995


 In article <pemanuel-0501950943340001 at beaker.ab.umd.edu>
 pemanuel at umabnet.ab.umd.edu (Peter) writes:

| I recently ran a 1% low melt gel isolation of a 700 bp PCR fragment. When
| I cut out the band and isolated it with GENECLEAN I found that a
| contaminating band of 1400 bp had gotten in there somehow. A friend said
| that can happen if you pverload the gel.  The top band piggybacks on the
| bottom because the gel is overloaded.  Load less was the recommendation. 

Eh? I'm having a very difficult time understanding this...the 1400 bp
fragment sticks to the 700 bp fragment while running through the gel?
I think your friend is mistaken, unless there is a big smear.

| If I load less then I must cut out a bigger band.  If you know anything
| about GENECLEAN you know that cutting out a bigger band spreads the
| isolations out over several tubes and can really lower your yield.

| So the questions are:
| 1.  Why is there a contaminant in there at all?

I think the problem may be that you've annealed the 700 bp fragment
to itself during the extraction process, perhaps by heating and cooling.
This happens when you digest lambda DNA with HindIII and run on a gel
without heating to 70 C first - there is a 12 bp overlap in the cos
site that must be melted in order to see the 4.4 bp fragment well.
Granted you're likely to have less overlap with a restriction digest,
this may be the same effect.
 
| 2. How can I avoid it? Or how much DNA can I load in a single well?

Try heating the extracted sample to 65-70 C before running it again on an
agarose gel. You may not see the contaminating band because it is 700 x 2,
in which case all is okay.

| 3. Would a lower percentage gel or running the gel a real long time avoid
| this problem?

Probably not. I say go ahead with the Genecleaned fragment.

| Thanks for your suggestions on this one.
| Peter

No problem.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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