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The Issue of Freezing and Using colonies of Tranformed DH5a

DH5a transformation freezing

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7 replies to this topic

#1 tihong10

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Posted 27 January 2013 - 05:55 PM

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 [amp] 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

#2 phage434

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Posted 27 January 2013 - 07:49 PM

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?

#3 tihong10

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Posted 27 January 2013 - 08:00 PM

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.

#4 bob1

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Posted 28 January 2013 - 12:40 AM

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

#5 tihong10

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Posted 28 January 2013 - 03:05 AM

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...

#6 phage434

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Posted 28 January 2013 - 06:01 AM

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

#7 bob1

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Posted 28 January 2013 - 11:38 AM

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.

#8 tihong10

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Posted 31 January 2013 - 02:15 AM

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!





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