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Help with bands resolution

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Help with bands resolution

Postby Dagon123 » Wed Apr 20, 2011 1:57 am

Hello,

I'm trying realize a subcloning. First, i digested 2ug of the vector with EcoRI to release the insert, the insert size is about 5,5kb and the backbone vector i'ts about 6,8Kb. When i runned the digestion into an agarose gel i saw only one band, and i need to purify only the 5,5kb band.
I'm using 1% of agarose concentration and running the gel to 100V for 2 hours. I also merge two wells of the gel to load the 2ug of digested DNA.

My question is, what is the correct agarose concentration and voltage that i have to use to resolve both bands? maybe i need to use lower agarose concentration and lower voltage too.. but,, what is the correct procedure?.
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Postby JackBean » Wed Apr 20, 2011 5:18 am

0.7% or 0.8% gel should be fine
http://www.methodbook.net/dna/agarogel.html
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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