gel extraction problem - (Sep/19/2005 )
hello
i excised my plasmid into two fragments. One is 2 Kb ,the other is about 10Kb. The 10 kb is that i need, so i did a gel extraction,but after the extraction, i did a gel again,no band occured .i am confused now ,see in someone can help me ?
What gel extraction kit are you using? 10 kb is quite large, maybe the size of your fragment is too big for your kit?
How much of your sample did you run on the gel? I usually use quite a bit of my gel extraction sample (15 ul) before I am able to see a band on the gel
you would rather use a matrix assisted kit instead of a column. There is more chance your column retain your plasmid than the matrix do not catch it
Hi! Is very difficult to purify the big fragments.....because, the current methods can break the same....for what you need the fragment? if you must to make subcloning, is not necesary to purify.....ligate all!! is more easy to make the screening of the colonies, and select the big retard!!!!
chau!
We routinely gel purify and recover fragments of that size and larger using Qiagen's gel extraction kit. Stay away from glass milk based kits for large fragments (if they're still out there -- it's been awhile since I used one).
i excised my plasmid into two fragments. One is 2 Kb ,the other is about 10Kb. The 10 kb is that i need, so i did a gel extraction,but after the extraction, i did a gel again,no band occured .i am confused now ,see in someone can help me ?
An easier way to purify large fragments is to run the DNA on a low melt agarose gel. You can then cut the band out and treat it with Gelase which degrades the agarose polymer. The DNA can then be precipitated with ethanol and used in ligations. Any residual agarose sugars will not effect the ligation reaction.