Overnight SURE cell culture in LB-carbenecillin
Posted 17 July 2009 - 11:34 AM
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
Posted 18 July 2009 - 05:11 PM
Career In Life Science
Posted 18 July 2009 - 06:19 PM
Maybe try using pBluescript as a positive control and see what you get... that's a good starting point in my opinion.
Posted 20 July 2009 - 03:51 AM
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.
Posted 20 July 2009 - 10:30 AM
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.