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i dont get colonies - (Sep/03/2007 )

hello all
i try to transform JM109 E.coli with pQE30 vector with 1500 bp insert and i dont get colonies ...could you kindly help me to solve this problem


We need more information. What are your controls? What happened to them? How did you do the digestion, ligation, transformation?


both insert and vector were digested with BamH1 and Hind III , purified and ligated.
transformation was cariied at by heat shock 40 s and plated on lb plates contain ampicillin.
in th same time a shorter insert of 400 bp was successful at the same conditions.
thanks in advance


What are your insert:vector ratios?

What ligation conditions did you use? What temperature, what kind of ligase buffer did you use?

Did you recover you cells in SOC for 30mins?

Did you dephosphorylate your vector? If so what are the conditions? How much vector was dephosphorylated with how much CIP for how long?

The insert, where did it come from? Is it a PCR product or cut from another plasmid. If PCR made, how many basepair of skirting around the restriction site did you use? 6bp? 5bp?

When you mean " dont get colonies " do you mean there are no colonies at all on the plate? Or that none of the colonies on your plate contain the desired plasmid?

Was a sucessful ligation conducted in your lab resently? Both T4 ligase and ligase buffer do not tolerate freze thaw cycles well and go off.

The devil is in the detail. Thus my reason to call this skill a black art. We need a whole lot more detail to actually identify a problem.