My competent cells are misguiding me - (Oct/20/2005 )

Hi all,

I have one strange problem regarding transformation after ligation, only positive control which contains supercoiled plasmid are able to give colonies of high efficiency rest all reactions added with ligase are completely blank.
Iam pretty sure regarding each component like restriction enzymes, ligase, compatibility of selection. Suppose if gene is toxic what kind of impact on DH5alpha with stringent vector like pET.
Could any of you kindly give me some suggestions.

Thank You

Regards,

Madhy

-Madhy-

your results indicates that your ligation does not work. Was it single digestion or double digestion case? Do u use alkaline phosphatase? check your ligation ratio? there may be just concatamerization of your insert. or your akaline phospahtase was not completely removed.
Please write in detail
Best wishes
amit

QUOTE (Madhy @ Oct 20 2005, 03:36 PM)

-Amit Kumar-

QUOTE (Madhy @ Oct 20 2005, 12:06 PM)

Theres few chance that l the leaky protein expression from pET will kill your bacteria, usually the promoter is only fully active upon stimulation with IPTG. I would rather think that it's a ligation transformation problem. remember that SC DNA is giving 100 times more colonies that any very good ligation so it is not really an indicator !

pesji

-pesji-

Actually, there is a basal level of expression of the T7 polymerase. If your gene is toxic to E. coli, you need to control for this with a plasmid like pLysS or pLysE (from Novagen; here:

QUOTE

In lambda-DE3 lysogens, the T7 RNA polymerase gene is under the control of the lacUV5 promoter. This allows some degree of transcription in the uninduced state and in the absence of further controls is suitable for expression of many genes whose products have innocuous effects on host cell growth. For more stringent control, hosts carrying either pLysS or pLysE are available. The pLys plasmids encode T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase, and thus reduces its ability to transcribe target genes in uninduced cells. pLysS hosts produce low amounts of T7 lysozyme, while pLysE hosts produce much more enzyme and, therefore, represent the most stringent control available in lambda-DE3 lysogens.

However, in less you have good reason to believe your gene is toxic, I'd look first to your ligation as the source of your problems. Can you give us further details about your restriction/ligation/cloning procedure?

-HomeBrew-