TA cloning - no colonies after transformation reaction (Apr/01/2005 )

I am using pGEM T easy vector system from promega for TA cloning and I am not getting the any colonies after transformation, though my competent cells are good. I am not able to understand where I am going wrong.
Troubleshoots from promega say that my competent cells may not b efficient. I am using DH5 alpha strain of E.coli and every time i prepare them fresh though its written everywhere to make the cells in advance and store them at -80 C.Does TBE used for the preparation of gels has some inhibitory effect on cloning?

If everything is done correctly about clonig steps then the problem may be emerges from the AT/GC content of your insert. As far as I know some competent cells can not menage to carry plasmids bearing some type of inserts because they degraded by the host. I think you should consider to try some other host then DH5 alpha such as E.coli SURE which is modified well to overcome these kind of problems. Good Luck!

can ube specific about which type of inserts some competent cells cannot bear? are u sure that E.coli SURE will be suitable for blue/white selection. wat bout TBE?

>can ube specific about which type of inserts some competent cells cannot bear?

You can easly clone the inserts those have irregular structures such as inverted repeats or secondary structures with E.coli SURE, because what SURE stands for is "Stop Unwanted Rearrangement Events". E.coli SURE cells doesent have the genes deleting or rearrenging DNA fragments having irregular structures. It is also "Restriction-negative" to allow cloning of methlyated DNA. Hope you are not going to ask what does inverted repeat or secondary structure mean...

> are u sure that E.coli SURE will be suitable for blue/white selection...

Yes I am sure. This strain contains a F’ episome which has the lacZ deltaM15 mutation, making it suitable for blue/white screening. Dont forget to use XGAL/IPTG though...

> wat bout TBE?
Dont think so...At least I have been using TBE and have no problems yet.

> " I am using DH5 alpha strain of E.coli and every time i prepare them fresh though its written everywhere to make the cells in advance and store them at -80 "
Try not to use'em freshly.

I am using pGEM T easy vector system from promega for TA cloning and I am not getting the any colonies after transformation, though my competent cells are good. I am not able to understand where I am going wrong.
  Troubleshoots from promega say that my competent cells may not b efficient. I am using DH5 alpha strain of E.coli and every time i prepare them fresh though its written everywhere to make the cells in advance and store them at -80 C.Does TBE used for the preparation of gels has some inhibitory effect on cloning?