5 replies to this topic

[ixsix]

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

[bob1]

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

[Rsm]

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

[Dr Teeth]

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.

[bob1]

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.

[Rsm]

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