Hello everyone, I am trying to express a recombinant proteins by using E.coli BL21. The plasmid contains Ampicillin resistant gene. The plasmid was transformed to the E.coli BL21 and more than a hundred colonies can grow on the agar plate with 50 ug/ml Ampicillin. However, when I transfer a single colony to the LB medium with 50 ug/ml Ampicillin, no bacterial growth observed. I tried to lower the concentration of Ampicillin in the LB medium to 3.125 ug/ml. No bacteria can survive as well. Also, I am quite sure the E.coli BL21 is not contaminated. How can I resolve the problem? Thanks.
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Resistant strains can grow on Ampicillin agar plate but cannot grow in LB medium
Started by Biophilic, Mar 20 2011 06:58 PM
6 replies to this topic
#2
Ameya P
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Posted 20 March 2011 - 09:49 PM
Probably the plate on which you plated your transformants was too old or dint contain Amp at all. So your lots of colonies are not actually transformants and wont grow on Amp plates.
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#3
pito
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Posted 21 March 2011 - 07:23 AM
gt_ameya is right,
another option might be that your needle (you use to put colonies from plate to lb) is too hot when you tranfser your colonies from the plate to the LB.
Other reasons might be that you made an error in calculating the amount of amp for the lb or for the plates.
Another reason might be that when you plate the transformants, you use to much liquid causing them to be able to grow, even if they do not contain the insert (they can survive because of the liquid forms a "shield" against the ab, eum; hard to explain whatI mean.. I hope you get it?)
another option might be that your needle (you use to put colonies from plate to lb) is too hot when you tranfser your colonies from the plate to the LB.
Other reasons might be that you made an error in calculating the amount of amp for the lb or for the plates.
Another reason might be that when you plate the transformants, you use to much liquid causing them to be able to grow, even if they do not contain the insert (they can survive because of the liquid forms a "shield" against the ab, eum; hard to explain whatI mean.. I hope you get it?)
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
#4
pDNA
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Posted 21 March 2011 - 09:46 AM
i think the problem can be solved easily be re-transforming your BL21 cells with your plasmid-stock.
try to check the amp-Plattes by doing a control transformation with e.g. pUC19 (this should be done anyway to check the competence of your cells).
If you do all of this ...i'm sure everything will be fine!
Good luck!
Regards,
p
try to check the amp-Plattes by doing a control transformation with e.g. pUC19 (this should be done anyway to check the competence of your cells).
If you do all of this ...i'm sure everything will be fine!
Good luck!
Regards,
p
pito, on 21 March 2011 - 07:23 AM, said:
gt_ameya is right,
another option might be that your needle (you use to put colonies from plate to lb) is too hot when you tranfser your colonies from the plate to the LB.
Other reasons might be that you made an error in calculating the amount of amp for the lb or for the plates.
Another reason might be that when you plate the transformants, you use to much liquid causing them to be able to grow, even if they do not contain the insert (they can survive because of the liquid forms a "shield" against the ab, eum; hard to explain whatI mean.. I hope you get it?)
another option might be that your needle (you use to put colonies from plate to lb) is too hot when you tranfser your colonies from the plate to the LB.
Other reasons might be that you made an error in calculating the amount of amp for the lb or for the plates.
Another reason might be that when you plate the transformants, you use to much liquid causing them to be able to grow, even if they do not contain the insert (they can survive because of the liquid forms a "shield" against the ab, eum; hard to explain whatI mean.. I hope you get it?)
#5
Biophilic
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Posted 22 March 2011 - 07:31 AM
Thank you all. I tried to extract the plasmid from the BL21 which were grown on the agar plate with 50 ug/ml ampicillin but no plasmid was obtained. The agar plate is fine. It is new and the concentration of ampicillin is right. I think the problem may due to the transformation efficiency because I am using LB medium for transformation. I am trying to use SOC medium instead to see if there is any improvement.
#6
pito
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Posted 22 March 2011 - 11:46 AM
Its not normal you get colonies on a plate with antibiotic while your cells cant survive this antibiotic.. They need that plasmid to survive. There might be something wrong with how you plate them or how you prepare those plates.
Or you do have a plasmid, but you are doing something wrong with extracting it.
How do you know there is no plasmid? You ran a gel or ?
Or you do have a plasmid, but you are doing something wrong with extracting it.
How do you know there is no plasmid? You ran a gel or ?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
#7
leelee
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Posted 22 March 2011 - 07:05 PM
You say you are sure you added the right amount of amp to your plates, but could you have added it when the agar was too hot?
