Please, some Tips for Maxiprep - (May/11/2006 )
you need to vortex very well the pellet before adding your buffers. But for 200ml i use 10ml of each buffer. so 25ml in your case.
So divide your pellet in 4 and do the extraction with 10ml of each. (2 is not enough for 2 reasons. First you need to ba able to add enough isopropanol for DNA precipitation
Second, the very big viscous particle made of chromosomal DNA will retain proportionnaly more plasmid than a smaller one.
Resuspend the pellet well. Then add the lysis buffer and make sure the everything is mixed well (everything should be blue now).
As fred suggested , add more of each buffer or split the bacterial pellet into 2 or 4 parts and do the maxi.
good luck !!!
Thanks a lot. That's good advice.Do you recommend me to use 4 tips to do this, or use the same tip with the 4 solutions? After all I don't get too much plasmid.
Other question: Before I used 100ml and I got only 45ug and again I think it was a problem at the beginning, with the mixture of P1, P2 and P3 and centrifugation. In this case, neither I got clumps nor colorless spots. What could I have done wrong that time?
well... I don't quite get what you mean by tip... except if it was pipett tip.
In you meant that, it's because i wasn't clear. I meant yesterday, do a big culture (assuming a 200ml culture), and pellet your bacterias with 4x50ml tubes. Than proceed with prep protocol.
Tough points : after pelleting cells : vortex very well the pellet. Add the P1 buffer and vortex well again (cells are better separated if no p1 added, or if you add P1, they are better suspended if you add a little volume of P1 for vortexing and add the rest of P1.
when adding P2, invert normally 8times. and keep on ice for 10-15'.
When adding P3, invert 8times and let sit 5' on ice.
If you don't get clumps, check the buffers, or prepare new ones.
for the tip, are you talking about the column? yes, I think you can probably add all your supernatants to the same column. this will increase your concentration and probably your yield as well
and Fred is so right, you need to vortex the holy bejeezus out of the pellet when you add the P1. you can't hurt it by doing this, and the solution must be completely homogeneous. sometimes small clumps can be broken up by drawing through a pasteur pipet several times if you are tired of vortexing and want to switch for a minute
So Fred, after you added P2, you incubate for 10-15' before adding P3? Qiagen states you shouldn't let the P2 incubation take too long?
Also: with the P3 buffer, the protocol states you should immediately transfer to the column and then incubate 10'?
Do your steps really help increasing yields (meaning: have you compared the yield with the normal protocol and with this altered protocol)? would be nice to get some info (and have higher yields).