3 replies to this topic

[Elvishswimmer]

Hi, I'm new to this forum.

I was reading around trying to figure out what is wrong with my bands. I'm in a undergrad genetics lab, and so I can't try and redo the gel, I just have to describe the things I did wrong.

Sorry for the crappy image, the paper is a bit curled: http://i9.photobucke...mmer/Photo3.jpg

My problem is the first 3 lanes after the ladder (1Kb). The dark blurs at the bottom, I don't know what it is from.

This is the specifics of the experiment:

0.8% agarose mini-gel.
visualized with Ethidium Bromide
buffer is 0.5X TBE
The gel was run at 80V for 1 hour and 10 minutes

The 3 lanes are all plasmids.
I digested Genomic V. fischeri DNA and pBluescript plasmid DNA. I then ligated them. I then transformed them into competent XL10 Gold.
I plated them onto LB/Amp/Xgal/IPTG and selected one isolated colony from a white colony, a blue colony, and a colony that had a successful lux transformation (was luminescent). I grew the 3 colonies in an overnight culture and then preformed a plasmid mini-prep.
So the first 3 lanes are undigested plasmids from the white, luminescent and blue cultures respectively.

I'm still new to the whole molecular bio labs, and am really unsure as to what I may have done wrong/forgot to do to cause those dark spots at the end of the gel.
There are also a bunch of other problems with the other lanes (for example, the empty lane! oops!) but I think I can explain them, its just those first 3 that I have no idea.

Any help would be appreciated! Thanks.

[phage434]

I would guess degraded RNA.  Is there RNAse in the first buffer used in your miniprep?

[NemaToStella]

RNA would be my guess, too. Looks familiar to me  

[Elvishswimmer]

Thanks guys! I was trying to go through my head and it seemed like RNA would be the only reason.

We did not use an RNase for those ones I think (don't have my notebook on me right now to say 100%)... some of the others we did.... that might have been the problem.
RNase wasn't provided for us for those first preps.... silly undergrad labs!

Thanks for your help

Edited by Elvishswimmer, 18 February 2010 - 10:42 PM.