plasmid minipreps

Helen Rolph hjr2f at udcf.gla.ac.uk
Tue Apr 13 06:24:56 EST 1999


I am using the  Topo TA cloning  kit from Invitrogen with the pCR 2.1
Topo vector to clone 16S rRNA gene fragments from mixed microbial
communities. The cloning efficiency appears to be quite high and I can
amplify the insert from the clones with no difficulty at all. 

However, when I try to obtain a high yield of clean plasmid DNA for dye
primer sequencing (LiCor method), the yields are very hit and miss. 
I have been using the Promega Wizard Plus kits to perform the minipreps
(with the modifications for endA positive strains). However, the yields
are very low (for example I recently obtained only 2 succussful
minipreps out of 14....the other clones had absolutely no DNA in them at
all, and yet I know they carry the plasmid with insert from previous
analyses). The week before, I had perfomed the minipreps on the same
clones and had onbtained plasmod DNA from all of them but at a very low
yield.

Any comments are really welcome

cheers


Helen Rolph



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