Trouble with viral vector --> too many colonies in control plate - Trouble with viral vector --> too many colonies in control plate (Jul/29/2007 )

Hello there,

i have problems with cloning gfp in my retroviral vector (12kb- RCAS) using Fermentas RE:

i inserted two cleavage sites into the vector (Cla1/Spe1. sequence analysis was positive. my cloning strategy is to directly clone the insert which has Cla1 and Spe1 compatible ends (gfp) into the Cla1/Spe1 opened RCAS vector. actually this should be a very easy to do cloning.

so i double digested the RCAS vector with Cla1 and Spe1 (the Cla1 site is about 20 nucleotides away from the Spe1 site: should this short distance of the cleavage sites make any problems?????). the linearized vector is about 12kb large. after 24h incubation with restriction enzymes Cla1 and Spe1 i made a gelanalysis and cut the 12kb band out of the gel and purified the Cla1/Spe1 double digested vector.
Next i ligated the Cla1/Spe1 restricted insert (GFP) into the RCAS vector.
For control i just pippeted the RCAS vector with ligase in a tube.

The next day i had hundreds of colonies on the control plate and also on the RCAS_GFP plate. Picking colonies doesnt make sense in this case.

I suppose that on of the enzymes did not cut and that the vector recirculized with ligase. But this seems a little bit confusing because digestion with Cla1 OR Spe1 showed, that these enzymes cut the RCAS vector (12kb). The plate contained Ampicillin. My suggestion that the antiobiotic selection did not work turned out to be false (plating e.coli wihtout Amp resistance did not grow on the plates)

Have you any suggestions??? How about dephosphorylation after double digestion???




regards.

I'd indeed try dephosphorylation, gel purification will not help you out in this case as single cut vector will not be separated on gel from double cut vector.

How did you prepare your insert? PCR or restriction from a vector?

Try digesting the vector DNA with both enzymes individually and run them on gel. This might suggest if one of the enzymes is not working at all or working only partially. This could explain the colonies in the control plate.

to go in the same way as scolix, i would suggest to add BSA in both digestions. It's not inhibitory and enhance the dispon,ibility of the enzyme for DNAcut.
Spe1 is slight tough. I would recommend an overnight-digestion try.

thanks. i already tried individual digestions and overnight inkubation.

both enzymes cut as i mentioned in my topic.

i posted a protocol for MCS digestion. Here is it again.
It has enhanced my clonings :
I've done tons of topoTA (same i think as pGEM easy and related, and gel purification is worse than digesting PCR reaction. i wish i could quote all the posts and members who have said avoid gel purif and prefer as much as possible pcr digestion.
anyway, do a PCR in digested pCR template, and digestion of PCR insert should be done 24h at 37 (it is not too much).

After that, for the vector try this protocol tha works better for me if it's a polylinker you want to digest :
i do 2 30µl-digestions in 2 tubes of 2µg plasmidwith the different enzymes.
Than i pull the tubes and add 0.5µl of each enzyme and go again for 3H.
I dephoshphorylate and column purify.
The idea background, is that there may be steric disturbing by enzymes. So the overnight digestion cut maybe 99% of 1st site, leaving the MCS alone for second enzme action.