3 replies to this topic

[kaveh]

Hi,

I have a pUAST plasmid in TE buffer obtained from a Maxi prep. I need to increase the concentration of the plasmid for getting a higher efficiency in downstream experiment (injection into Drosophila embryo). What would you recommend?

1) can I do an ethanol precipitation and recover the DNA in a lower volume? Is that a problem with the TE buffer?
2) speed vac?

thanks

Kaveh

[Papaver]

I always used speed vac to concentrate my plasmid. But before you should check that the saltconcentration is in normal rate otherwise it would be to "salty" after speed vac. And you should think about the final concentration. Is it realisitic to get your desired amount after speed vac?

[phage434]

You can definitely re-precipitate from TE.  Add 1/10 volume of sodium acetate pH 5.2, then 2.5 volumes of cold ethanol, cool for 30 minutes at -80, spin down hard, wash with 70% ethanol, dry (but not too much, just until you can't smell the ethanol any more) and resuspend.  If you have trouble with visualizing the pellet, add 1 ul of Novagen pellet paint NF before you start.

[virusfan]

If I am not mistaken, for microinjection, it is prefer to have your DNA in molecular grade water, not TE buffer.