7 replies to this topic

[emadsadeghian]

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?

[Pangea]

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

[phage434]

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.

[emadsadeghian]

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.

[bob1]

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.

[emadsadeghian]

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.

[Pangea]

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?

[emadsadeghian]

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