Minipreps and Automated Sequencing

Mr. O.Coutelle NIMR olli at crc.ac.uk
Sat Feb 4 18:37:57 EST 1995


In article <3gujs2$kov at lyra.csx.cam.ac.uk>, cdw1000 at bioc.cam.ac.uk (Christopher Walentas) writes:
> Does anyone out there know what the best way to prepare low-copy number plasmid
> (mini-prep scale) for ABI automated sequencing?  We've tried Promega Wizards
> (Magic uesd to work but that's another story), but they don't like to yield
> any sequence even with additional precipitations/ washed to remove excess salt.
> We tried QIAGEN 20 tips-- they work great down the hall where they use pUC
> and cosmids, but the yields for pKK223-3 and pGP1-2 derived vectors are
> deplorable.  Folk here have even put CsCl banded DNA down a 20 tip to find 
> that they are only able to recover 10% or so.  What we've settled on is either doing larger minpreps (or rather a bunch of 'em), or chloramphenicol.  Is there a better way?
> 
> As an aside, if Promega was in patent conflict with their Magic resin, who 
> DOES have the patent so that I can go back to what used to work.
> 
> Please forward any and all suggestion to the email address below.
> 
> Cheers in advance!
> 
> 
> Tripp
> 
> - 
> Christopher D. Walentas
> 
> Centre for Molecular Recognition,		Tel.:
> Sir William Hardy Bldg.,			within the U.K.  (0223) 333662
> Department of Biochemistry,			outside       +44 (223) 333662
> University of Cambridge,			FAX		        333345
> Tennis Court Road, 				Answerphone & alt. FAX  333661
> Cambridge, England  CB2 1QW

I have frequently used miniprep DNA for taq terminator cycle sequencing on the ABI, with no problems
whatsoever. I found that standard alkaline minipreps worked as well as Wizzard preps, and I get reads
in excess of 350bp. I use about 1 ug of miniprep DNA directly in the sequenceing reaction. I have
also got excellent results with quiagen midiprep cosmid DNA, which I use to walk on with custom
made walking primers. There was virtually no noise, and I could get up to 450bp.
It seems to me, that the problem you have lies somewhere else. You could also PCR amplify your
plasmid inserts, and directly sequence the PCR products after PEG ppt with a nested primer, this
always works for me.

Oliver Coutelle
email: olli at med.cam.ac.uk



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