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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

-ixsix-

dry it until the ethanol is gone. You could try heating at 65 deg C for 5 minutes to help dissolve the DNA.

-bob1-

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

-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 4C 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.

-Dr Teeth-

Minna on May 20 2009, 10:00 PM said:

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


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.

-bob1-

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

-Minna-