uncut vector contamination in gel purified band - (Nov/15/2010 )

Hello all,

in the last two weeks I am experiencing a molecular cloning trouble that seems to be quite difficult to deal with.

I am trying to replace the HygroR cassette within the MSCV vector with a mcherry cassette. In order to do that I amplyfied the mcherry sequence from another vector and subcloned it in a pGMT after additing A nucleotides to the PCR purified product. Until here everything worked well.

The trouble started when I tried to replace the two sequence. I efficiently digested both pgmt vector and mscv to excide the mcherry and Hygro R sequence respectively without any odd bands. But unfortunately when I transform DH5alpha cells with the products of the ligation between the mcherry sequence and the MSCV I get almost 100% colonies containing UNCUT or eventually religated pgmt vector.

That seems to me particularly strange because I purify the mcherry band after gel separation so teorically no uncut or linearized pgmt vector should run at the same level of the mcherry sequence.

Do you have any explanation for that or any solution to rescue me from this situation?

Many thanks

I do not know if it could be of any help but the mcherry fragment is 1.1 kb in lenght and surrounded by a ClaI and AgeI RSs. I digested both the vector for 1 hour at 37C and I never saw any trace of uncut bands.

I performed ligation for 1 hour at RT. Before ligation I dephosphorilated the MSCV vector and this step is quite successfull as I didn't find any colonies on the control plates with the DH5alpha cells transformed just with the MSCV.

In contrast EVERY TIME I see an high number of colonies (~30) on the control plates transformed with with the mcherry fragment without any MSCV. The number of those colonie is approximately the same of the colonies produced after transformation with the ligation products (and in fact almost all the colonies after transfection contain the uncut pgmt).

how do you do the digestion?
Do u cut 1ug at a time?
or couple of ug at the same time
If you do that sometime you could get someuncut vector that you would end up ligation