I have Xl1 blue competent cells with a cat no. 200249 at -80C. in order to revive it (to make bacterial culture), wire loop were inserted into the culture(competent cells) and streak onto tetracycline containing LB agar media. The working conclusion of tet was 10ug/ml. culture was incubated at 37C for 15 hours, but upon observal there was no growth. please discuss it.
XL1 Blue cells are indeed tetracycline resistant. Check the manual from Agilent: http://www.chem-agilent.com/pdf/strata/200249.pdf
Innoculate atleast 100ul from glycerol stock or 10ul from prepared competent cells first in simple LB and let it incubate for 1 hour at 37. If any growth observe than add antibiotic.. or u may use it without adding antibiotic.
try this way, use a sterile pipette tip to touch the vial(sterile again!) and inoculate that tip in 3-5ml LB+Tetracycline mini culture, incubate it for the growth. This will ensure the proper inoculation (may be u missed in the loop while streaking) and make 2-3 independent minis, one without antibiotics, second with ug/ml tel and the last with 50ug/ml.
It should grow even in tet concentration 50ug/ml, in the next day, u can pick a loopfull of culture from this, and streak for single colony isolation if you want.
If nothing works, call the company and ask them do send a new vial.
Good luck.
As Shehzaib Siddiqui suggested, the frozen bacterial cells sometimes fails to growth in LB plus antibiotic. First strike the cells in a LB agar plate and growth overnight to 37C. Next day pick a single colony and inoculate LB+tet broth and/or strike another LB+tet agar plate.
Thanks for all of your kind responses,
My Xl1 blue competent cells has grown successfully,, let me share it.
after the first day when it didnot grow on tetracyclin containing media,
second day i spread 60 ul on two tet containg media and 50 ul on LB Agar media without antibiotic, and o/n incubate it at 37C, XL1 blue cells Growth was observed on LB agar media without antibiotic, but again not on tet containing media.
then i just pick single colony from antibiotic negative plate and streak on tetracyclin containing media, it showed growth on it, and also in LB broth containing tetracylcin antibiotic.
so it was interesting, it might indicate that
1. some of the cells loss plasmid during competency, while some bacteria still have. As they showed growth later on tetracyclin containing media, after their growth on simple LB agar.
2. plasmid were there, but requires some time on antibiotic negative media to code for their proteins, (as in this case tetracyclin also).
Anyway thanks once again.....
Shabbir khan..
Hi Shabbir,
I think that your second possibility is the most logical. Sometimes the protein that provide resistant to the antibiotic is in periplasm so it needs to be transported there.
should the concentration of tetracyclin used to grow XL1 be lower that normal?
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