Gel Electrophoresis problem

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BrianScott
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Gel Electrophoresis problem

Post by BrianScott » Wed Aug 27, 2008 8:27 pm

I'm sorry if I missed this topic being discussed before, but I did do a search and it produced nothing related to the issue.

Well the issues is when I'm running gels the plasmid DNA is sticking to the wells. I'm attaching an image of a 1% agarose gel I ran. I loaded two different samples of the pET vector in the two wells below the ladder and below that is the same pET vector (from the well above it) cut with EcoRI.
Image
I hope the image isn't to big, lol.

Both pET vectors come from overnight cultures I've worked up using Promega's Wizard Plus SV Minipreps DNA Purification kit. The thing is, is I'm starting to see this with more of my plasmid dna.

What I could use help on is advice on what might be causing the dna to stick to the wells like that.

blcr11
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Re: Gel Electrophoresis problem

Post by blcr11 » Thu Aug 28, 2008 9:19 pm

I've never used this kit, so I don't have a good feel for it. I can't see the picture of the gel, either. My guess would be that there is protein contamination that precipitates in the wells and traps the DNA. What is the A260/A280 ratio of your samples? If it isn't 1.7 or higher you might want to do the protease reaction either for longer or at a higher concentration of protease. You could also try a phenol/CIAA extraction or two followed by either ethanol or isopropanol precipitation, or passing it over another tube of resin.

hilim
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Re: Gel Electrophoresis problem

Post by hilim » Wed Sep 17, 2008 10:40 pm

i guess the mini didnwt work for you, what you see stuck might be gdna, anyway dont forget pet are low copy you should try harder for getting good amount of dna
:shock:
if you want to search other miniprep(as i remember i used promega and didnt have any problem) i have useful link for you that will help u searching pruduct
http://www.ibio.co.il/ProductsMatch.asp ... Kits+(Mini) :roll:

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