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Contamination of Ecoli.


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#1 emma999

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Posted 02 September 2004 - 08:38 AM

Hello

Can anybody help me. I am cloning a targeting construct using TOP10F' Ecoli. It was all going fine then suddenly I started to get back unusual DNA from my minipreps. This DNA is not related to my backbone vectors and does not digest with any enzyme. I have no colonies on my negative control plate - which suggests that my sterile technique is OK. The colonies on my positive plate look normal but once I grow up a 2 ml overnight the culture looks strange. I was thinking it could be phage contamination?
Please, any suggestions would be much appreciated. I have been cloning for around 6 years with no problems until now.

The DNA I get back runs in 3 bands on an agarose gel - which are always the same.

Best wishes
Emma
UK

#2 tuckern

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Posted 12 October 2004 - 05:14 AM

What size are yer bands - three bands like that sounds a bit like RNA!!

#3 emma999

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Posted 12 October 2004 - 07:25 AM

Dear Tuckern, thanks for the suggestion.

The bands were not RNA as I RNAse treated the preps before digestion. I have never found out what they were but I changed the SOC and LB which I use after electroporation and everything is working now - the bands have disapeared! The SOC is made for the whole department so I think it was contaminated with some weird plasmid DNA that took over my cultures. Luckily it is now gone and I am not going to ever use the communal solutions again. Word of advice if things are going wrong check the reagents you haven't made yourself.

Emma




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