I'm trying to separate human allelic bands by gel electrophoresis, and these fragments are between 150-400 bp long. What concentration agarose gel would I need to resolve these bands? I am making a comparison between different peoples DNA, so I need to seperate the bands to an extent where I can differentiate between the different people.
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#2
hobglobin
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Posted 29 January 2011 - 11:33 AM
I'd try frist a 1.5-2% Gel and lithium borate buffer (instead of TAE/TBE), a sufficient long gel is necessary too. But I guess you'll never reach the resolution of a sequencer i.e. a long polyacrylamid gel.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
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Baileys
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Posted 29 January 2011 - 12:12 PM
hobglobin, on 29 January 2011 - 11:33 AM, said:
I'd try frist a 1.5-2% Gel and lithium borate buffer (instead of TAE/TBE), a sufficient long gel is necessary too. But I guess you'll never reach the resolution of a sequencer i.e. a long polyacrylamid gel.
Hi hobglobin,
Thanks for your response. I'm yet to try the lithium borate buffer. Have tried TAE and sodium boric acid (as seen in Brody and Kern, 2004), but lithium boric buffer is my next step
What length gel would you recommend? Do you think a 12 cm gel will do it, or would a 20 cm be better?
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hobglobin
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Posted 29 January 2011 - 12:17 PM
Baileys, on 29 January 2011 - 12:12 PM, said:
hobglobin, on 29 January 2011 - 11:33 AM, said:
I'd try frist a 1.5-2% Gel and lithium borate buffer (instead of TAE/TBE), a sufficient long gel is necessary too. But I guess you'll never reach the resolution of a sequencer i.e. a long polyacrylamid gel.
Hi hobglobin,
Thanks for your response. I'm yet to try the lithium borate buffer. Have tried TAE and sodium boric acid (as seen in Brody and Kern, 2004), but lithium boric buffer is my next step
What length gel would you recommend? Do you think a 12 cm gel will do it, or would a 20 cm be better?
The longer, the better I'd say.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
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Ameya P
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Posted 01 February 2011 - 01:04 AM
Baileys, on 29 January 2011 - 12:12 PM, said:
I'm yet to try the lithium borate buffer. Have tried TAE and sodium boric acid (as seen in Brody and Kern, 2004), but lithium boric buffer is my next step
Would just like to add that TBE buffer is better at resolving smaller fragments. A 2.5% gel in TBE separates bands 20-30 bp apart for me after 50mins of electrophoresis.
Hope that helps!!!
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