The Issue of Freezing and Using colonies of Tranformed DH5a - (Jan/27/2013 )

Hello all,

I am attempting to obtain a plate of DH5a tranformed with a plasmid, but I am having issues with the antibiotic concentration. Non-transformed DH5a control grew on LB agar plates with AMP concentrations of 50 and 100 ug/ml. Therefore I am currently experimenting with the AMP concentration to find the amount that will prevent non-transformed growth. If I had unlimited resources (i.e. ligation mixture) I would simply perform a fresh transformation to test different concentrations of ampicillin; however, I do not. I have on hand the following: (1) a week old transformation mixture (frozen immediately after completing the transformation procedure) (2) about a week old plate of colonies of transformed DH5a that grew on that allowed non-transformed DH5a growth (i.e. 50/100ug/ml) and (3) a plate of control DH5a that arose from the plating of frozen DH5a control (i.e. a tube of DH5a that underwent the transformation process without addition of plasmid).

I want to find the ideal amp concentration without wasting limited ligation mixture and competent cells but also without using a bacteria source that may be compromised. My biggest concern is with the frozen transformation mixture. Can I assume that using a transformation mixture that was frozen immediately after the transformation process is approximately equivalent to using a freshly prepared batch of transformed DH5a in terms of genetic integrity?

And lastly, can I assume that using a colony of plated non-transformed DH5a is approximately the same as using non-transformed DH5a from a freshly prepared batch of control DH5a (having undergone transformation without a plasmid). Will the DH5a be the same in both cases? I have also been told that leaving a plate to incubate for more than 1-2 days maybe lead to arise of mutant strains; is this true?

Thank you,
tihong10

Untransformed DH5a should definitely not grow on 50 or 100 ug/ml agar plates. Something is wrong. Are you waiting for your agar to cool to 55 before adding the anitbiotic?

Yes, made the first batch of 50ug/ml plates by adding antibiotics when I could fairly comfortably keep my hands on the flask of LB. When untransformed DH5a sprang up I took more quantitative measures and sat the LB flasks in a 53 deg water bath for 30 minutes before adding ampicillin. Even so, I found that the DH5a grew on both 50 and 100ug/ml plates. So I thought either somethings wrong with the ampicillin or I am using too low of a concentration of it. A fellow lab member doesn't believe there is anything wrong with the ampicillin, and the competent cells were purchased from a supplier and kept in a freezer so the only option I had remaining was the amp concentration.

30 min may not be long enough from autoclaving or microwaving - try an hour or two.

I think I will try a 2hr water bath but I am quite certain the temperature was correct. I actually water bathed a total of about an hour. The temperature leveled at 53 deg for about 30 min, and when I added amp the flask was not even mildly uncomfortable to hold indefinitely...

More likely is contamination of your cells from some source. Water baths used for heat shock often contain amp resistant bacteria, for example.

Ok, in that case, make up some fresh stocks of Ampicillin and/or check that your bacteria do not already have an Amp resistant insert - which is the only other possibility. DH5a should not be Amp resistant without either the Amp being off or having a resistance gene inserted.

I am remaking my plates and using a new source of ampicillin and a 2hr water bath - hopefully the results come out better than the first time.

Thanks bob1 and phage434!