pTopo purification problems

cjmcdermottroe from gmail.com via methods%40net.bio.net (by cjmcdermottroe from googlemail.com)
Sun Sep 13 12:45:47 EST 2009


Everybody has already suggested the obvious - dodgy amp/plasmid prep kit etc. I clone into topo more often that i'd like and dont bother with blue/white screening. Instead, i just use a no insert control plate as an indicator. Typically (for an average sized insert at a v:i ratio of 1:3 and ligation time of 5 min) i'll get approx 20 and 150 colonies on the no ins and the 1:3 plate, respectively. you ought to concentrate (again as already mentioned) on establishing whether or not your amp is off. Transform some bugs with a preprepared amp resistance plasmid that you or a colleague has in the lab. These should grow on amp agar whereas untransformed bugs should not. If this is the case, ensure the prep kit is ok by preping a colony or 2 from the above plate containing bugs that you transformed with your amp plasmid. Stick it on a gel (preferably linearised) and if you have a band, all is good. If not, buy a new miniprep kit.  

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-original message-
Subject: pTopo purification problems
From: "Yoram Gerchman" <gerchman from research.haifa.ac.il>
Date: 10/09/2009 10:15 pm


Greetings netters
I have recently made an attempt to clone a short gene into pTopo (using Top10
competent cells). i did get white colonies on LB-Amp-X-gal, and they grew
nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried
already 2 different kits). Any ideas as to how remediate this situation?
Many thanks
Yoram



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