DNA purification loss, large insert

[Michael L. Sullivan]

>
>
>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 
your ligation.

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
>agents?

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.

Mike

Michael L. Sullivan
Plant Physiologist
US Dairy Forage Research Center
Madison, WI 53706


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