Dear all,
I've try to purify the digested plasmid (16 kb) from the agarose gel using the gel purification kit. Anyway, I got very low concentration of the plasmid after purification. And I cannot use it in my ligation reaction.
I also did my insert (200 bp) purify as control but it's fine.
So I don't know how to fix this problem.Please anyone suggests me.
Many Thanks
Pannapa Pinweha
Dear Pannapa
You said that you got very low amount of digested plasmid after purifying from agarose gel, is my understanding correct? How you determine the concentration before purifying from the gel. For me, to handle with large DNA is quite tough, be make sure that you gently handling the sample during your purification process. Shear force somehow damage your DNA and it cannot be detected if it become small pieces. I don't think that you have DNase in the condition since you told that for the small piece DNA was fine for you. Not vigorously vortex mixing.
I don't know it just my view , if you can solve the problem please let me know too.
Good luck :)
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