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Overnight SURE cell culture in LB-carbenecillin


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#1 Ms new scientist

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Posted 17 July 2009 - 11:34 AM

Hi Guys! I am new to this forum but I was hoping you can help me.

I have been trying to clone a gene into SURE cells using Pmal vector. I did the ligation and transformed the plasmid into SURe cells. I got growth on my transformation plates (about 2 colonies in each plate) so I inoculated 5ml LB broth with Carbenecillin (for selection) with one colony and grew the cells overnight. But the next day, my tubes dont go cloudy and when I centrifuge the tubes to get the lysates (as per QIAspin mini prep protocol), I dont get any cell lysates at the bottom of my tubes.

Can anyone help me please?? I have tried this transformation and inoculation for over 2 weeks now and still no result!

Thank you so much

#2 bochum

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Posted 18 July 2009 - 05:11 PM

When you facing such problem always use a positive control (Hope you got a positive control plasmid along with the kit).If not use the plasmid which already worked in your lab as positive control. And when you are not getting a enough cells then it not worth to go further with DNA preparation so first sort out first problem then go further. Any way i am not experience with SURE cells but 2 colonies per plate sound strange i dont know whether it is normal numbers...check it out with the company or with your seniours in your lab...
With Regards
Martin
Career In Life Science

#3 LordPhantom

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Posted 18 July 2009 - 06:19 PM

What concentration of antibiotic are you using? It might be too high, which is why you are only seeing 2 colonies. I would recommend doing positive, negative, and background controls.

Maybe try using pBluescript as a positive control and see what you get... that's a good starting point in my opinion.

#4 altobarn

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Posted 20 July 2009 - 03:51 AM

I faced the same problem for getting too few colonies but those colonies never got insert in it when I perform test digestions.

My advice is clone the fragments into TOPO cloning first and subsequently subclone it into your own vector. By doing this you would get higher concentration of DNA insert compare from the fragments you got from PCR.

#5 Ms new scientist

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Posted 20 July 2009 - 10:30 AM

Thank you so much for your help.

I asked a few seniors in my lab and they said two colonies is good enough for my type of coloning but it is strange that when I take the colonies for overnight growth in Carbenecillin-LB, they do not grow and the solution does not go cloudy. Even when I am able to isolate plasmids, the concentration I get from mini spin prep kit is so low (25ng/ul).

Oh and my Carbenecillin concentration is 50 ug/ml.

Thanks again




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