co-transformation - (Aug/09/2011 )

hi
dose anybody know how can I make it efficient to transform 3 compatible plasmids they are:
RSFDuet-1(k+)
pETDuet-1 (A+)
pACYCDuet-1 (cm+)
my inserted fragments are about 1kb and less,
yesterday I have transferred all but today there is no colony there,
also would you please tell me that usually how long would it take for the colonies to grow?
also another question;
how much is the concentration of the hygromycin for e-coli selection,
thanks
mary

Easiest is to transform one plasmid at a time. Start with (say) the RSF one, select on Kan. Take those cells, transform with pET, select on Kan + Amp, then take those cells and transform with pACYC and select on all three antibiotics. You should not need high transformation efficiency if you are transforming with purified plasmid, so a CaCl2 transformation will probably work well. Else, an electroporation is easy.

thanks alot for your answer, would you please help me in finding the cacl2 method?
also I want the co-expression of my plasmids, so it doesn't if I transfer them step by step?
also after each step I should just take the e-coli and add the other plasmid by the heat shock method?
if there is any protocol would you mind sending me,
regards

m_rajaee on Wed Aug 10 12:37:05 2011 said:

You should be able to find a protocol yourself, but here is a start: http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf
(you can find protocols in papers too.. so maybe you need to look for papers in your field or papers that are also working with your plasmids)


You transfer them step by step, so the co-expression only starts after step 2 , when you tranferred the second plasmid.

And yes, each step is a step on itself, so yeah, you do 3 tranfers , 3 transformations , 1 for each plasmid.