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double transformation problem


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#1 Cristinaaaa

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Posted 23 May 2013 - 11:41 PM

Hello, everyone.

I am doing a double transformation, of E. coli (BL21), with two plasmids, one with chloramphenicol resistance, and the other one with ampicillin resistance. Before starting the experiment, I checked that the plasmids are compatible. After I make the transformation (with 1 µL of each plasmid), and grew overnight on fresh DYT Cm10/Amp100 plates, I got nice singe colonies. Then I picked single colonies in 3 mL DYT Cm10/Amp100, and I get no culture after 18 hours. What could be the problem with my colonies?

Thank you.

Have an great day!
Cristina.

#2 phage434

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Posted 24 May 2013 - 05:01 AM

I don't know what is wrong -- probably destruction of amp on the plates. I'd strongly suggest that you transform one plasmid at a time. You might also switch from ampicillin to carbenicillin, which is much more stable.

#3 notjustalabtech

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Posted 09 June 2013 - 11:43 AM

Like what Phage434 says, it's possible the amp is degrading on your plates. Make sure you pick colonies without colonies around them and which are on the outside of the "smear". Even if the amp is stable, it's possible there are so many colonies on the plate that the amp is being "eaten" or used by the faster growing stronger colonies, allowing non-amp resistant ones to grow around them. You could also reduce the overnight incubation time to 14-16 hours, and the first ones to grow will be the amp resistant colonies.

#4 Cristinaaaa

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Posted 11 June 2013 - 05:19 AM

Thank you very much for your replies. I tried another strand of BL21, the BL21-CodonPlus® (DE3)-RIL-X, and I could culture them, and did the expression of my proteins. Could this effect be because of the fact that one of the plasmid/protein is toxic to the initial strand of BL21?

Thank you again and have a great day!
Cristina.




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