Hi everybody,
I am trying to digest a couple of reporter plasmids and having some problems with the size of the expected bands. I enclosed a photo of my lastagarose gel. The first lane is a pGL3 plasmid containing LTR-luc reporter nondigested (size 7446 bp), the second one the plasmid digested with Eco RI (band sizes 2722 and4724 and the tird one the same plasmid digested with PST I (4431, 5389 and 958 expected bands). Then after the ladderthere is another undigested plasmid (PNMT-luc reporter) whose size should be 5533 bp, followed by thedigested one with Not I + Xba I (bands 2625 and 2908). First problem is I cannotfigure out why the gel runs so badly. The run is done at 70 V for 3 hours, thegel is 0.7 % using 0.5% TBE as buffer. The main problem though is that thebands are not the size expected, with some of them even bigger thanthe undigested plasmids. The ladder is an Invitrogen 1 Kb DNA ladder. The digestion is done at 37 C for 1h and 15mins, using in this case 0.8 ug of plasmid DNA.
Does someone know what the problem could be?
Thanks very much.
Annalisa
Gel_Digestion18feb11.ppt
