DNA purification loss, large insert
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Sat Apr 13 10:00:43 EST 2002
>I'm considering ligating them in the gel slices, using low melting agarose.
>Does anyone have a reliable method for extracting DNA frags over 5kb that
>gives an acceptable recovery yield?
Low melt agarose can work well for this. Run your insert on a 0.8% gel in
0.5X TBE and excise the band in as small a piece of the low melt agarose as
you can. Make sure you are using a long wave UV source for this so you
don't nuke your DNA. Weigh the agarose slice, and add 3 ul H2O per mg of
gel. Melt at 65 degrees and run a few ul on a gel along with a standard of
some sort to get a rough concentration. With such a big insert, you
shouldn't have much trouble getting a high enough yield to use directly in
Alternatively, somebody recently gave me a protocol for extracting
fragments from regular agarose. Essentially the gel fragment is frozen in
liquid nitrogen and then micorfuged. The liquid that is released from the
agarose is supposed to have most of the DNA. This can then be used
directly, or precipitated and resuspended in a smaller volume for use. I
haven't tried this method yet, but plan to next week. I have a more
detailed protocol I can provide, but I don't have it on my computer at home
where I am now.
I think your ligatioin should be doable. I don't think one really needs a
great deal of DNA to make the sort of construct you've described.
>Also, any suggestions on the ligation of such large pieces? Should I bother
>with a rapid ligation kit, or use plain old T4 ligase without crowding
I've never used a rapid ligation kit. "Regular" ligation (I like to use a
formulation with PEG-- essentially the 5X ligation buffer of BRL) is plenty
rapid for most applications IMHO, and I sometimes make pretty big
constructs for plant transformation. One suggestion for you, though. Your
final construct is going to be plenty big. If you're not transforming by
electroporation, you might consider that. My understanding is that
transformation efficiency drops off considerably for large plasmids using
chemically competent cells. Efficiency with with large plasmids is
apparently not such an important issue with electrocompetent cells.
I hope these suggestions help.
Michael L. Sullivan
US Dairy Forage Research Center
Madison, WI 53706
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