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J-an's Content

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#61784 PFGE conditions

Posted by J-an on 08 March 2010 - 11:55 PM in General Lab Techniques

Hi,
I would like to ask if somebody can recommend me switch times to separate fragments from 20-250 kb using CHEFII aparature.
In manual it is recommended to run 0,05-10 sec puls during 2-15 hr to separate 1-100kb sizes, howewer i need to separate bigger fragments too. I use 0,5 x TBE buffer and 1% agarose concentration.
i already tried to combine 7-20sec for 12 hours + 20-50sec for another 12 hours and i found my fragments low separated on the bottom of the gel,
then i tried 0,5- 6sec for 6 h + 10-35sec for 16h and i got little better result but i also found my fragments on the bottom of the gel.
i also tried to tun 10 hr 1-6 sec + 5 h 7-20sec and this time i got my fragments in centre of the gel but they were also low separated. i want to split them up through entire gel.

Does anybody have a suggestion how to successfully separate my fragments?

thx




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