midi-prep - (May/20/2009 )
Hi everyone,
I meet a problem when extracting plasmid.
It is the last step, dissolve the plasmid in TE beffer. After air dry for 30 min (probably too long?), I can still see the white pellet in the tube. Then I add 100 ul TE buffer, but the pellet does not dissolve! I collect it into a new tube anyway, and votex it and leave it at room temperature for 1 hour. But still the plasmid pellet does not dissolve.
Could anyone give me any suggestion?
Thank you and have a very nice day!
Dan
dry it until the ethanol is gone. You could try heating at 65 deg C for 5 minutes to help dissolve the DNA.
30mins should be enough to get rid of the EtOH. Anyway, from a 70% EtOH solution, the EtOH should vaporize quicker than H2O, so remaining liquid is more likely to be H2O (or did I sleep during chemistry class again?).
Maybe your yield is too high? If you're using ion exchange kits, you`ll get around 500ug plasmid. You won`t be able to dissolve that much in 100ul H2O, better use 200ul. Furthermore, I would suggest you to pipet the pellet up and down to mix, instead of vortexing...
Cheers,
Minna
It is possible to overdry the pellet, making it difficult to dissolve. Air drying for 5 min should be plenty assuming you have aspirated out the visible 70% ethanol wash. Once the pellet is dry, it should be relatively clear-- a white pellet after drying often indicates salt or protein contamination. My advice is spin down your pellet, wash several times with 70% ethanol and completely aspirate each time. Then, resuspend in TE and let sit at 4°C overnight. In the morning, gently vortex, centrifuge and transfer to a new tube. Test your 260/280 ratio by spectrophotometry and run some out on an agarose gel to confirm.
Minna on May 20 2009, 10:00 PM said:
Maybe your yield is too high? If you're using ion exchange kits, you`ll get around 500ug plasmid. You won`t be able to dissolve that much in 100ul H2O, better use 200ul. Furthermore, I would suggest you to pipet the pellet up and down to mix, instead of vortexing...
Cheers,
Minna
You don't get left with a pool of water on your bench after wiping down with 70% - they should evaporate equally more or less.
You're right...
DaveC426913 says: "Although the boiling point of ethanol, 78.3 degC, is significantly lower than the boiling point of water, 100 degC, these materials cannot be separated completely by distillation. <...> This means they both evaporate mostly together...."
Strange world we live in...
Cheers,
Minna