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Trouble cloning E.coli cells with pUC18 and insert

electroporation competent MKH13

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#1 Biol74

Biol74

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Posted 31 January 2012 - 09:51 AM

Hi all,

I hope someone can help. I am currently having trouble cloning 7 PCR products (between 1.5kb and 2.5kb) into E. coli MKH13 using the pUC18 plasmid (2686bp). I use positive and negative controls during transformaion (i.e electroporate just the plasmid (no insert) and electroporate just the cells without plasmid). The PCR products show good strong bands on a gel after purification so I know this is not the problem however there are many other steps to consider when trying to find the source of the error as outlined below.

I was getting no growth on any plates when using LB with 100ug/ml Ampicillin. I have dropped the Ampicillin concentration to 50ug/ml as the literature suggests that this concentration of Ampicillin should suffice. However now I am getting growth on LB with 50ug/ml but when I tried extracting the plasmid I found there was no plasmid on the gel suggesting that perhaps the wild type was growing at 50ug/ml. I did an MIC on E.coli MKH13 also and got an MIC value of 1ug/ml suggesting that 50ug/ml would be more than enough to inihibit the growth of this organism without the plasmid and insert.

Also I am making the cells electrocompetent myself (using the glycerol wash method) as I don't think you can buy competent MKH13. I am strictly following this method and ensuring that the cells remain cold at each stage however i'm wondering if the electroporation transficiency is playing a role. I have plated the electrocompetent cells on LB and got a high colony count (TNTC at 10-5) however although there is a large number of cells it does not necessarily say that the cells are electrocompetent so I will need to electroporate the cells with plasmid and do a count again. Does anyone know if you can buy electrocompetent MKH13 if this doesn't work?

Apologies for the essay! Hope someone can shed some light on this! I read on this forum today that greater than 4ul of ligation mix can actually inhibit transformation. I was using 5ul of ligation mix and 50ul of competent cells so I will try it with 2ul ligation mix next. Any other suggestions/comments would be much appreciated :)





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