Hi everyone,

I am digesting 1 ug of pmiR-glo vector with Xhoi and SacI restriction enzymes.

When I started to minimize my volume (50 to 30) I started to get strange bands. Can this be a result of start activity?

When I digest with only 1 restriction enzyme SacI HF (NEB) I got correct linerized vector.

I got these bands only when I reduced the volume in to 30 ul.

my reaction setup is;

1 ul xhoi

1ul saci

0,2 ul of 5ug/ul plasmid

3 ul cutsmart

23 ul autoclaved dH2O

for 4 hours

Normally;

1 ul of each enzymes

3 ul of 5ug/ul plasmid

5 ul cutsmart

40 ul dH2O

four overnight

but in this digestion my vector linerized without any insertion. I got 5-10 colonies when I perform ligation on digested vector. So I thought that some of the plasmids are digested by only 1 enzyme due to the fact that I overly used plasmid DNA than I are ligated again. So I reduced the plasmid consantration per reaction 15 ug to 1 ug but I also reduced reaction volume.

Does these bands can be a result of star activity due to the reduced volume of reaction?

Best Regards