pTopo purification problems

Bari Zahedi via methods%40net.bio.net (by bzahedi from bccrc.ca)
Thu Sep 10 18:48:19 EST 2009


This is very little info. It could be your bugs, your lysis, your amp is off, it could be many things. Can you provide more info. 

To narrow down if the problem is at the plasmid prep level then: 
1)To start, did you also get blue colonies? Can you get any plasmid out of those? This will help determine if there is something wrong with your kits/plasmid prep reagents. 
2)Do you get anything coming out of your preps? Like a smear? If so the problem maybe with your lysis.


-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Yoram Gerchman
Sent: Thursday, September 10, 2009 12:55 PM
To: methods from oat.bio.indiana.edu
Subject: pTopo purification problems


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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