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transformation fails, bacteria grows in control plate

ampicillin transformation

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6 replies to this topic

#1 Summer July

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Posted 16 August 2011 - 12:04 AM

I made a mutant, and transform the DNA (prepared from miniprep) into competent DH5a, the construct should be ampicillin resistant but even on the control plate (where I didn't add any DNA to the competent cells) were growing full...
I was thinking whether the antibiotic doesn't work any more but my colleague uses the same batch and it works just fine. Any idea what happened here? is it because of the cells?
Thanks for help!!

#2 Summer July

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Posted 16 August 2011 - 12:44 AM

any suggestions please????Posted Image

#3 GNANA

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Posted 16 August 2011 - 01:59 AM

your cells might be contaminated.. either during transformation or during the preparation itself (if at all it is home made comp cells)..., better change the cells and do it....you will probably get an answer ....

I always had an alternate hypothesis....


#4 Summer July

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Posted 16 August 2011 - 02:21 AM

your cells might be contaminated.. either during transformation or during the preparation itself (if at all it is home made comp cells)..., better change the cells and do it....you will probably get an answer ....

Thanks! But I have been doing a lot of transformations, I never made such a contamination (On the control plate, it is really full of bacteria after over night incubation). And these competent cells , which are home made, I have been using them for a long time, never had a single problem...

#5 GNANA

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Posted 16 August 2011 - 03:23 AM

Do a sterility test before proceeding any experimental transformations from that batch....

I always had an alternate hypothesis....


#6 phage434

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Posted 16 August 2011 - 03:55 AM

Grow a sterile plate, and streak some known strains with and without amp resistance on some of your plates (you could do this on one plate).

#7 Summer July

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Posted 16 August 2011 - 03:57 AM

Grow a sterile plate, and streak some known strains with and without amp resistance on some of your plates (you could do this on one plate).

Do a sterility test before proceeding any experimental transformations from that batch....

Thanks!! I am doing it now, so far I don't see any problem with the antibiotic or the cells. Because in the control, bacteria is growing while in the cultures with amp, nothing is growing...





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