in response to bolcsodal, the plasmid has a pUC origin and an f1 origin ... is this what you mean? is a 1500 or 2600 bp insert to big for a 5.6 kb vector?
in response to relaxin, yes the T7 primer site is close to the cloning site (like 10 bp away). the thing is - the insert is supposed to be in the multiple cloning site but the T7 primer sequencing data don't show base pairs until like site ~3300 of the vector.
The exact vector I'm using is pcDNA3.1/Hygro(+) from Invitrogen.
The WEIRD thing is that the sequencing data (using T7 primer) for the 1500 and 2600 inserted constructs show that the beginning of the sequence starts right after that SV40 pA site - at the EXACT spot for both of them. You'd think that if it were random cutting due to the comp cells or whatnot, I should get random sized vectors with no insert then. But 4 clones screened from the 1500 bp constructed vector and 4 from the 2600 bp constructed vector plates give different bands from each other, but the same within the 4 clones themselves (sorry, don't know how to say that more clearly). Again, if it were random, I shouldn't be getting the same result within the same ligation/transformation reaction right?
So from the sequencing data - it seems like the Hygro resistance, SV40 pA (i don't even know what that is), the SV40 ori, f1 ori and BGH pA should all be cut out. Is this a correct assumption?
I've also done the PCR test for the inserts - got nothing. The only one that worked is that 65 base pair one. Sequencing data came back and it was in there and PCR analysis of the construct showed the insert was in there as well.
I'm stumped and so is my supervisor!