Xho1/Bglll digest of pSUPER.retro.puro with stuffer problems - (Sep/04/2008 )
I am having a hard time digesting this vector, I first did a double digest with promega enzymes for one hour at 37 C. I got a stuffer band at around 1kb but the supposedly double digested vector was about 60 times more than the other band. The band was really think and it didnt really look like everything had been double digested.
Then I did a sequentiel digest using Xho1 form Rosch and and Bgl2 from fermentas. I digested with each for 2.5hours in a special buffer called RB 100. Again the result was the same except for the fact that I got two stuffer bands at 1000 and 900 somehow.
What else can I try? Was digesting 10 and 5 micrograms of vector a mistake? Other enzyme companies like NEB?
Please give me some ideas!!
What is the size of your plasmid that you are digesting and what size fragment are you expecting?
When loading gel, try to load not more than 1ug (better 500ng) per well, it can help visualize things better.