20bp ladder doesnot open fully even in 4% NuSieve - (Jan/16/2008 )
Hello everyone.
I am working with 20bp to resolve small restriction fragments (20 to 170 bp) on 3% agarose and 4% Nusieve. However on both i am only barely able to read the ladder. I am running these gels at 100 volts for 30-35 minutes. Could this be the problem? Is it too much? By the way i have tried running all the way. The ladder only moves as an whole and does not open
What about loading less ladder?
I am working with 20bp to resolve small restriction fragments (20 to 170 bp) on 3% agarose and 4% Nusieve. However on both i am only barely able to read the ladder. I am running these gels at 100 volts for 30-35 minutes. Could this be the problem? Is it too much? By the way i have tried running all the way. The ladder only moves as an whole and does not open
I'd try running everything at a much lower voltage and for longer. You should see better band separation.
Has the ladder been through repeated freeze/thaw cycles? You may try heating the ladder at 65C for about 10min and see if that helps.
100V doesn't tell us anything without the distance between positive and negative pole. What conditions does the provider of your ladder suggest?
btw: check this topic, might help also! http://www.protocol-online.org/forums/inde...showtopic=33389
I am working with 20bp to resolve small restriction fragments (20 to 170 bp) on 3% agarose and 4% Nusieve. However on both i am only barely able to read the ladder. I am running these gels at 100 volts for 30-35 minutes. Could this be the problem? Is it too much? By the way i have tried running all the way. The ladder only moves as an whole and does not open
I routinely use 20bp ladder on 15cm, 3% Agarose 1000 (Invitogen) or 3% MetaPhor (Lonza) in 0.5X TBE with no problems. I run at 150V for about 4-5 hours - this runs bromophenol blue and fragments below 100bp right off the gel so if I wanted the smaller fragments to stay on I'd run for 2.5 - 3 hours maybe.
The company which has supplied the ladder shows a picture in the literature acconpanying the ladder in which the ladder was run at 5V/cm for one hour on a 10 cm gel. does this mean i should run it at 5*10=50 volts for one hour
I am working with 20bp to resolve small restriction fragments (20 to 170 bp) on 3% agarose and 4% Nusieve. However on both i am only barely able to read the ladder. I am running these gels at 100 volts for 30-35 minutes. Could this be the problem? Is it too much? By the way i have tried running all the way. The ladder only moves as an whole and does not open
I routinely use 20bp ladder on 15cm, 3% Agarose 1000 (Invitogen) or 3% MetaPhor (Lonza) in 0.5X TBE with no problems. I run at 150V for about 4-5 hours - this runs bromophenol blue and fragments below 100bp right off the gel so if I wanted the smaller fragments to stay on I'd run for 2.5 - 3 hours maybe.
ok i'l try running it for 2-3 hours. We are used to running our 1.5% agarose gels at 100V for 30 minutes!
By the way Isn't 150V too high. Some students have complined of their gels melting at high voltages
Very interesting. do you suggest i keep my ladder at RT or in the fridge rather than freezer (-20) I had the impression heating it would damage it somehow
I am working with 20bp to resolve small restriction fragments (20 to 170 bp) on 3% agarose and 4% Nusieve. However on both i am only barely able to read the ladder. I am running these gels at 100 volts for 30-35 minutes. Could this be the problem? Is it too much? By the way i have tried running all the way. The ladder only moves as an whole and does not open
tried that. same results