Problems with plasmid digestion

[annalisa80]

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

[CharonY]

First regarding Gel quality: 3h is usually awfully long. What kind of gel is it? Minigels should not take longer than about 30 mins. With a longer run you can have buffer depletion effects if the reservoir is not large enough. This often results in smears in the high-MW areas. This appears to have happened in your case as the low MW bands look better. What is the total volume of running buffer?

Also I assume you used 0.5x TBE (i.e. 1:10 dilution of a 5x stock) and not 0.5% TBE?

 

Regarding undigested plasmids: the size assessment is only accurate for linear DNA as the confirmation (CCC, OC, linear, etc,) affects electrophoretic mobility. Therefore undigested, non-sheared plasmids will always run faster than linear DNA of similar size. However, I cannot really tell you why the linear fragments do not fit the sizes, though.

 

First thing I would do is to verify the correct electrophoresis conditions (including correct buffer composition and strength). For such a long run either use more volume or maybe try up to 1x solution. After you verified that the under the given condition at least the ladder looks perfect, start worrying about the fragment sizes.