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pGEX-KG Vector insert in BL21 Codon Plus RP, Growth Difficulty

RP Codon Plus BL21

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#1 razasohail28@yahoo.com

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Posted 02 June 2013 - 11:13 PM

Hello Dear ALL
Hoping you will be fine. i am using BL21 Codon Plus RP for expression of my protein using pGEX-KG Vector. Because pGEX-KG is Ampicillin Resistant so i use Ampicillin+Chloramphenicole Plates to culture the bacteria but when i transfer the colonies in Ampicillin resistant LB broth or the broth which contain Ampicillin+Chloramphenicole then the colonies not grow in broth. Colonies only grow in Cloramphnicole resistant broth. Then i have checked my plasmid(GENE) using PCR but all colonies are negative (colony which grow on Chloramphnicole resistant broth). Please guide me in this issue
Thanks

#2 Curtis

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Posted 03 June 2013 - 01:34 AM

We had a similar problem last time. It was because the amp in our plates already degraded. We had a lot of colonies on the plate, but they didn't grow in broth. Now we prepare fresh amp plates every month.

#3 razasohail28@yahoo.com

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Posted 03 June 2013 - 06:04 AM

I have already tried new plates but problem remain same

#4 Missle

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Posted 03 June 2013 - 01:48 PM

The fact that they are growing in the presence of chloramphenicol is the same as if regular BL21 (DE3) cells were growing in LB with no antibiotics but not growing with ampicillin. The chloramphenicol is there to maintain the rare codon tRNA-containing plasmid in the BL21 (DE3) Codon Plus host strain. If the colonies grew on the ampicillin/chloramphenicol plates and then can not grow in the presence of ampicillin then you are loosing the plasmid (or something is wrong with your plates - wrong ampicillin concentration, agar too hot when amp added, etc - and the plasmid was never there in the first place). If you're trying to PCR amplify your gene from purified plasmid, your plasmid is likely the Codon Plus plasmid and not your pGEX-KG vector.
Have you had success with other plasmids / host strains using your methods & media?

#5 razasohail28@yahoo.com

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Posted 04 June 2013 - 12:44 AM

Yes i have success using other E.coli on same batch ampicillin plates





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