Purification of 13 kb plasmid from E. coli - (Jun/06/2007 )
I want to do maxiprep to purify my 13 kb plasmid from E. coli. I had a problem when I was doing minipreps with spincolumns. Since they had a binding capacity of 10 kb!!!
does anybody have an idea how I could purify it or do the maxi columns have a more binding capacity?
Thank you for your ideas,
how about basic alkaline lysis? This trial and tested method does any plasmid from 3kb to 200kb.
However if I do recall correctly somebody has mentioned binding columns which can bind >10kb. Unfortunately I am unable to find that post and thus verify and provide a product name.
I would try the spin columns. Heat the elution buffer and let it sit on the column for 5 minutes before spinning it down. You might elute twice this way.
Qiaex II will purify fragments quite a bit larger than 10 Kb.
I use Qiagen kits (miniprep, midi and maxi) on a regular basis for purifying large plasmids (up to 15 kb): no problems so far. I do heat the elution buffers, but apart from that I just folow the protocol. Probably kits from other kits will work too.
Yes, heat the EB buffer is the key.