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Determining ideal page conditions


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5 replies to this topic

#1 andrewpickering

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Posted 30 August 2009 - 10:05 AM

Hello

So I'm trying to seperate on a gel a 700kDa band from a 2000kDa band.
under my standard conditions (12% gel, 150v, 1-1.5hr) this obviously doesn't really happen.
I was wondering which conditions I should change and by about how much?

#2 bob1

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Posted 30 August 2009 - 04:13 PM

lower your gel percentage, adjust time/voltage as necessary.

#3 andrewpickering

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Posted 30 August 2009 - 04:38 PM

lower your gel percentage, adjust time/voltage as necessary.


Yeah I figured that, I was more hoping for a kind of idea how far to adjust.
i.e. is 8% gel sufficent or should I go lower?
should I run it for 2hr --> 3hr --> 4hr --> 6hr?

#4 phage434

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Posted 30 August 2009 - 06:26 PM

Lower percentage. You could run a prestained marker with high molecular weight, and you would be able to visualize when the marker was in the correct position. The buffer used will also affect the speed of running. Invitrogen has a good page on gel buffer compositions, percentages, speed, and sells prestained markers (along with many others such as NEB or Pierce).

#5 K.B.

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Posted 31 August 2009 - 07:34 AM

I would try somewhere around 5-6%. Keep in mind that gel at this percentage will be very soft.

Gradient gels (eg. 4-10%) would be nice, if you can afford them...

Run until bromophenol blue is few mm from the bottom of gel.



For this molecular weight range size exclusion chromatography would be much better than PAGE.

#6 bob1

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Posted 31 August 2009 - 01:55 PM

Try also Roche Lab FAQs for a really useful set of basic guidelines for many things in the lab.




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