transformation inhibition due to gel extraction?
nico.dantuma at mtc.ki.se
Thu Nov 13 12:36:00 EST 1997
The last few weeks I have been struggling with some persistent cloning
problems. All the obvious steps (digestions, dephosphorylation and
ligation) were checked and no problems were observed. Yet, even after
transforming bacteria with ligation in which ligated products were clearly
visible on an agarose gel, no transformants were obtained. Encoding of a
toxic protein by the recombinant plasmid is not the reason for this
failure. The latest experiment pointed in the direction of the insert
which, according to this preliminary result, also inhibits the
transformation of bacteria with intact vector.
The insert was isolated using the Qiaex gel extraction kit. I have used
this kit in the past routinely without any problems. Recently, I observed
that after the elution a minor amount of the buffer becomes viscous and
remains attached to the Qiaex carrier. Despite the los of these few
microliters the yield was acceptable. I am quite sure that this was not the
case in the past. I thought that this was due to centrifuging at 4oC which
I until then always performed at room temperature. However if the eluate
only contains elution buffer + DNA this never should become viscous. Other
reasons could be 1) I am using different brand of agarose and 2) the kit
was modified with an pH indicator
Does this problem sound familiar to one of you and more important what can
I do to overcome this problem?
Thanks in advance.
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