Why restriction digestion fallout after combining the size is more than the vector plus insert
Answers
I have been using HindIII-HF and XhoI as my double digest enzymes and hints point to an unbalanced digestion.
Background:
Currently struggling with constructing a reporter plasmid using my target insert and several different mammalian vectors: pcDNA3.1(+), pDsRed-Monomer-C1, pEGFP-C1, pGEX-3X. I have two different Insert sizes, 1.7 kb and 3k bp.
Try constructing the plasmid using classic restriction digest with two restriction enzymes. They should be placed well in the vectors as well. Leave 6 bases overhang in the primers, so that the endonucleases could cut properly. So, the general primer design is no the problem, since after PCR see bands at the correct size on the gel. Cut out gel slices, purified them from the gel and used the DNA in the restriction digest for the insert. Linearised the vectors using the same procedure. Linearised vectors showed the correct size on the gel as well.
Hope It Helps!!!!????,