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Methods for concentrating plasmid DNA - (May/09/2006 )

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you 'd better centrifugate your plasmid solvent before you getting concentrated .then useing the 30ul solution resolve again .which is very what you need the concentrated plasmid with 30ul solution


QUOTE (T. reesei @ Dec 2 2006, 05:18 PM)
hi fred what is the recipie of the butanol???
any dilution ?? or u add some other reagents??/

ouuuuuuuuuuuuuuuuuups unsure.gif unsure.gif unsure.gif i missed that one...
well butanol is commercial solution so the "recipe" is quite easy.
Procedure :

add at least 10 volumes of butanol. Vortex well if you can add supplemental butanol, add it and revortex.
if a lot of bubbles remains in the solution, a 5' spin at top speed is sufficent.
Pippett the upper phase (caution : it's an organic solvent and should be properly watsed)
repeat the porcedure as mentionned.
if you don't see pellet, rego for a 10' spin.

the basic behind this :
butanol is very hydrophobic. So if bubbles are in large amounts, DNA easily goes in it and the pelleting is easy. upperbubbles act like carriers as, when the reach a lower bublle, fusion occurs and the resulting bubble is bigger and pellet better.
in the last steps, when butnaol is in sufficent amount, bubbles may be too small at the beginning. So they need little more time.
At final step, when the solution appears clear, the DNA is in small particles. So a 10' spin alows to recover all DNA.

procedure (continuing)
wash 3 times with EtOH 70%

advantages : for small volumes, it's amazing because you need "only" 10' and you get quite all the material in your solution. (better than etOH and IprOH
in quite all cases you have better recovery.
For RNA pelleting, you add in that way only 1 solution, inappropriate for RNases and i don"t lke salts solutions as powder are often unsure in a lab where all people use them. (so are the magnetic stirrers)


i don"t lke salts solutions as powder are often unsure in a lab where all people use them. (so are the magnetic stirrers)

so what to you do? do you have your own magnetic stirrer??
usually when i work with some important sample, like for RNA i bake the stirrer for 8 hours in 180c

-T. reesei-


how could I precipitate DNA that is soluble in ddH2O without any precipitation procedures?
Isn't it possible to spin down the dna (15'000 rpm / 20-30 min / 4-10°C) and then use the speed vac to decrease the volume?
Here I wouldn't lose so much dna, or?


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