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Can't isolate plasmid from cloned E.coli

cloning TOPO Cloning kit

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4 replies to this topic

#1 ElizabethJM

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Posted 26 September 2014 - 05:46 AM

Hello, please help me. I did cloning using TOPO TA cloning kit Dual Promoter. I grow the E.coli on LB + amp agar and broth, and it show significant growth. But when I tried to isolate the plasmid using miniprep kit and alkaline lysis, the gel show no band. What should I do? BTW, I'm cloning beta lactamase gene in the vector. 



#2 Bio-Lad

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Posted 26 September 2014 - 06:05 AM

Well, assuming that your ligation worked and that your cells transformed correctly (since they are Amp resistant), I'd say there is a problem with your plasmid prepping.  What kind of kit or protocol are you using?

Edited by Bio-Lad, 26 September 2014 - 02:18 PM.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#3 phage434

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Posted 26 September 2014 - 07:03 AM

You should make sure that your original cells are not also ampicillin resistant. Ampicillin is not particularly stable, so if you have growth after a long time (days) then the cells may not be resistant. Also, your plates might not be prepared correctly. It would make sense to do a control growth of your "transformed" strain against the original cells on the same plates to make sure that the plates are actually selective. I would also, in future, switch to carrbenicillin as an antibiotic, which is more stable and superior in every way to ampicillin.



#4 labtastic

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Posted 18 October 2014 - 05:46 AM

 I would also, in future, switch to carrbenicillin as an antibiotic, which is more stable and superior in every way to ampicillin.

 

Except for cost...carb is almost 10x's the price.

 

Unless you know a vendor I don't, in which case please do share.  :)



#5 ibesky

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Posted 21 April 2015 - 06:10 AM

If you're in the states, try RPI Corp for Carb. Worth every penny. We switched over 2 years ago and haven't looked back since.







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