Transforming DH5 alpha cells with pUC19 vector - Cloning (May/23/2005 )

Hi,
I transformed 100 microliters of DH5 alpha cells with 1 microgram of uncut pUC19 vector and plated the cells on LB plates with Ampicillin. I was expecting Blue colonies but all I am getting are White colonies. I used fresh antibiotic but got the same results. I had made the competent cells myself using the Z competent kit. I am wondering if there is a problem with my competent cells. Is there any other way to check for efficiency of the competent cells. I used X-Gal from Promega (used 40microliters of a 2% w/v solution per plate). Any suggestions would be welcome.
Thanks

Hi
I think there is no problem in ur competent cells but the concentration of plasmid u r using for transformation is too high. 100 ul competent cells need only 50ng of plasmid for transformation. the higher the concentration of vector is used for transformation the decrease in efficiency of transformation occurs. still if u want to check the efficiency of comp cells u can plate them on Amp+ and Amp_ plates. If u get no growth on Amp+ plates then only ur comp cells are efficient for transformation and are not contaminated, but I will suggest u to dilute ur plasmid before doing transformation.
Good luck[seema]

Hi,

Don't you also have to add IPTG to media when using DH5alpha? Or am I wrong?

Freiberger

also is there an insert in your vector disrupting the lac gene and therefore you have lost lac expression and thus no blue colonies?

Nick