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Gel Electrophoresis band problems


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#1 Elvishswimmer

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Posted 18 February 2010 - 02:12 PM

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.

#2 phage434

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Posted 18 February 2010 - 03:46 PM

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

#3 NemaToStella

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Posted 18 February 2010 - 05:41 PM

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

#4 Elvishswimmer

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Posted 18 February 2010 - 10:40 PM

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 :P

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





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