Troublshooting failed transformation - pLEASE hELP (Feb/19/2009 )

hI aLL GEEKS,

I have done transformation using MC1061/P3 ultracomp E.COLI with tetracyline (10ug/ml) ampicillin (25ug/ml).
I did teh positive control as well. I used diffirent dilutions of DNA, diffrent medium like NZYME AND SOC, but none of them worked.
Everything was used fresh, like plates, antibiotics and medium etc. New tube of competent cells was opened.

Can anyone suggest something what could be teh possible reason?

Do you think my competent cells are no longer competent though they have been purchased just last year in august from invitrogen and since then kept them in -80C FREEZER.

If you did not get any thing from your positive control, you have to check whether your competent cells are OK, and your transformation was properly done (right temperature...) and your plates have the right concentration of antibiotics, your incubator is at the right temp.

by the way, what plasmid is pLEASE ?

pcrman on Feb 20 2009, 03:15 PM said:

To check teh competency of competent cells, i transformed with competent cells from some other department too and again that failed. Every thing was right i guess, 30 minutes incubation, heat shcok for 40 seconds at 42C , medias, receommended antibiotic concentration.

The plasmids is human GABAA alpha2 subunit cloned in to pcDM8 vector.

try transforming without antibiotics, maybe your concentration of antibiotics is lethal

pcrboy on Feb 21 2009, 06:21 PM said:

Do you think that will give me my desired plasmids? Won,t there be a chance of contamination?

Did you allow a growth period (say 30 minutes at 37C with shaking) after transformation but before plating? This is required with tetracycline due to phenotypic lag, or your transformants will die. You might also consider dropping the tetracycline to 5 ug/ml; some resistance genes don't convey resistance to 10 ug/ml. Why is your ampicillin concentration so low? I usually select for ampicillin resistance with 100 ug/ml...

HomeBrew on Feb 21 2009, 06:00 PM said:

Yes, i allowed 1 hour incubation at 37 Co before plating.
I have used the antibiotics concentration recommended by invitrogen for MC1061/P3 chemically competent cells.
I cant see any growth at all on the plates.

I have tried using the glycerol stock as well for growth in LB medium using tehsame antiviotic concentration but no growth.

looks like everything is coming out negative, try something that is considered a positive control. which is why i suggested growing without antibiotics.

competent cells should stay fine at -80, so that isnt my major concern. reason is you're assuming your bacteria has recovered from competency while at -80...which suggest they have undergone a lot of growth. try growing untransformed competent cells and transformed cells in no antibiotic media. let's see whats positive and whats negative.

pcrboy on Feb 21 2009, 07:38 PM said:

Thanks PCRBOY for your kind suggestion. I will try that without antibiotics too but i have tried the positive control using ampicilline only plates according to teh recommendations of the invitrogen but that did not work too.

Now i think teh only option is to grow them wd no antibiotic plates.

the media you grow them in should also have no antibiotics. also, i wouldnt screen bacteria that has been grown in normal media, reason is because as you mentioned earlier, there is high risk of contamination.