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my competent cell grow on any agar plate


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#1 emadsadeghian

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Posted 04 January 2013 - 04:50 AM

I have been given a batch of XL1 blue cells. I made them competent using calcium chloride procedure. To test them, i plated them out on agar plate containing: Kanamycin, Tetracyclin, Ampicilin and control. After overnight incubation, there is bacteria colonies in all plates. These colonies are so dense we cant not see them separately.
Since I have kept my original stock of cells before making them competent, I did the same test on the original stack and i saw colonies only on the tetracyclin plates (XL1 Blue is resistant to Tet). This means the procedure of producing the competent cell has somehow made my Ecoli strand a multiresistant strain.
My question is if this is possible and if not why this has happened to my batch?


#2 Pangea

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Posted 04 January 2013 - 05:27 AM

Are you sure that your agar plates are alright.? Do you have positive and negative controls.? And check your protocol doing the competent cells.

#3 phage434

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Posted 04 January 2013 - 06:07 AM

Another possibility is that you have contaminated the cells during your competent cell prep with something other than E. coli. Tap water or ice often contains resistant organisms.

#4 emadsadeghian

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Posted 04 January 2013 - 06:52 AM

Pangea, yes, all plates even those with no antibiotics and those with tet+kan showed growth,
Phage434, thanks for letting me know the source of this problem. probably ice or water contained these resistant organism

Edited by emadsadeghian, 04 January 2013 - 06:53 AM.


#5 bob1

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Posted 05 January 2013 - 03:01 PM

Were they fresh plates?- could easily be that your antibiotics are old or no longer working, this could especially be the case if the agar was too hot when the antibiotics were added.

#6 emadsadeghian

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Posted 07 January 2013 - 06:28 AM

hey guys,
i carried out the experimental procedure again to make new competent cell from new stock of xl1 blue. I tested them with right antibiotics. They only grew on Tet and control (o antibiotic), It is great news. Its because I autoclaved my equipments before use and I made sure the whole procedure is absolute sterile eliminating the contamination problems.

#7 Pangea

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Posted 07 January 2013 - 07:32 AM

Under which condition are you growing the stock solution cells before transforming with plasmid to have enought cells? Or are you using a stock aliqout?

#8 emadsadeghian

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Posted 08 January 2013 - 04:25 AM

we have aliquots of XL1 blue, i spread them on agar plate containing tet, i took out a colony, did a 15ml starter culture, then did the 500ml culture, i centrifuged out the pellet and using calcium chloride, i made the cells competent. Then I tested them on agar plates containing antibiotics, they worked perfectly.
Therefore i did not do any transformation. I just did normal testing on the cells




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