DNA molecular weight keeps changes - (Sep/24/2010 )

I've been having problem with my DNA not running at the correct MW. I did gel extractions for my vector and insert after restriction digestion. If you look at gel purify.jpg, the removed gel slice with my vector is on the left of the marker and that containing my insert is on the right. My digested vector runs at ~8kb according to the ladder when it's actually 4.2kb. My insert looks ok at ~3.2kb. However, after gel extraction, even my insert is running high at ~5kb (gel purified vector insert.jpg)! I run the uncut vector and it's at the right MW. Has anyone experienced this?

What could be wrong? Not only are the MW changing, the higher MW bands are poorly resolved/smearing. I'm using Fermentas 1kb GeneRuler. Instead of using 1uL (500ng), I premixed the loading dye and ladder such that I use only 1/5 the recommended amount (100ng). Could premixing be the problem (eg dye interference)? I've done this at my last lab with NEB 1kb ladder with no problem. I tried a new tube of ladder and it's still the same. But then, I used SB and ethidium bromide there instead of TAE and GelStar. Stains are added to the gel. I've tried preparing new buffers from commercial 10x TAE and making new gels. I'm running at 100V (~5V/cm). Is that too high?

Please help. Thanks!

Adding EtBr to the gel retards electrophoresis such that MW is no longer accurate IIRC.

But shouldn't my DNA and the ladder be affected equally? The difference is just too great, isn't it?

The ladder and the plasmid could be affected differently if there are conformation issues. It could also be that you have a lot of salt in the preparation, especially after gel extraction. This may affect how the DNA runs