help separate 2 DNA bands of only 100 bp difference and elution ! - already tried 2% but some thing wrong help!! (Aug/15/2006 )
After restriction digestion i will get two bands . but they have only 100 bp difference
i have to separate the 2.5 kb ( with insert )and 2.6 kb rest of vector in agarose gel .
after separation and elution i have to clone that 2.5 kb into another vector .
i tried 2% agarose gel, but the bands doesnt seem to separate
i added the ethidium bromide to the dna sample , does it affect the dna mobilty??
the gel size is not much in my lab,,, so that restriction is there, so cant make long gels and run it for long time ,,
i ran it at 100v .. for 25 min ,, in 1X TAE
how to separate ?
please help soon
try PAGE for separation. 10% i'd say. You should be able to easily resolve that difference
Use a lower percentage of your gel if you need agarose. Go down to 0,6-0,7% and let your gel run slowly for a long time.
I agree with Vairus, lower percentage and let migrate for a long time.
Run it at 40-50V for a longer time, 1hr-2hrs. And use a lower % gel as others suggested.
I agree, use 6 % agarosa gel. You shouldn’t have problem to separate them.
I had to clone in an expression vector some 3 kb insert cloned already in a 3 kb vector T. What I did was: cut with the suitable RE to free the insert and at the same time, cut with a RE inside the vector. Then, I had 3 kb band (insert) and 2 smaller bands (vector).
Last time, I just purify the entire 3 kb band (mix of inset and vector), I cloned, and picked 10 colonies and looked for a clone with the right insert (4 clones had it).
thank you all
i will try agarose scine i need to do elution afterwards
aztech princess ,
thats was a nice idea ,, i wonder why i didnt think of that
i wil try that frist
thanks you all again
See if there's another site in the vector that isn't in your insert, and digest with it. Your plasmid will go to smaller fragments, but your insert will still be happily sitting at 2.5 kb.
That's what i was just going to say.
I had a 3kb product cloned into pBluescript (3kb), and the only way to seperate it was to find a enzyme that cut the pBluescript but not my insert. It works really well, and it's a lot less fuss than making special gels.