Bugs grow on plates but not in media - (Sep/03/2004 )

My cloning issue is that my transformed E coli grows on LB/kanamycin plates but once I pick the colonies they do not grow in LB/kanamycin broth. I did not get that many transformed cells (about 80 in all). The vector is the TA cloning vector into TOP10 cells. The vector is also ampicillin resistent as well but they did not grow in LB/ampicillin either.

My thoughts are:

1. The kanamycin plates do not have active kanamycin and I do not really have transformed cells.

2. Make new LB/kanamycin plates and broth and try again....

Does anyone have any other suggestions as to what may be going on?

As far as I can remember that TOPO TA is blue white screening, Am I right?

if that is the case, go for the white colonies (at least after 24 hr incubation)
then grow them for 24 hrs in fresh 50 ug/ml ampicillin in LB broth, then isolate the plasmid and verify that you have your insert in the right frame by sequencing.

as far as the kanamycin, I suggest that you check the concentration of your kana stock. then make new LB-Kan plates and LB-kan broth. I had used 30 ug/ml successfully in the past for protein expression in pET-vectors.

It is always important to include positive control and negative control with an experiment like that. You never know what is going to happen.

You can do the following:
-Run a pos ctlr for the plates to verify that the kanamycin is working
on the plate (cells without vector and insert), you expect no growth

if your kanamycin is working.
- plate the transformants on kanamycin plates you expect growth (this is what you want).
- Before plating, make sure you shake your cells in (250 ul)SOC medium for 1 hr at 37 C at 250 rpm to let the cells express their resistance genes and maximize the transformation efficiency (Check your transformation protocol).

you can also do controls for the transformation protocol.

I hope this is useful for you.

Good Luck
Mbiologist 1

If the insert is toxic, growing the cells at 37C may force the segregation of plasmid-free cells. Try growing the cultures at 25C to 30C instead.

Hi,

I agree with mbiologistI. You have to make controls pos and neg.
We dont take TOPO but TA Dual Promoter but I think thats not the problem.
for the transformation maybe take dh5alpha competent cells (Invitrogen). With your cells we have former times also problems.
Take new solutions at all as MBiologist says
-overnight culture with LB (4ml) with 4ul Amp or Kan (50ug/ml). Take the white colonies with a white tip and just in the tube with LB, 37C° 200rpm shaking, next morning miniprep (Qiagen), colonie PCR with Uni and Reverse Primer for the pCR2.1 vector, sequencing.

Its just standard protocol but maybe it can help you

Good Luck

ein Leidensgenosse

Thank you so much for the suggestions. Will try variations of these