4 replies to this topic

[arnabde2000]

After transformation and innoculation for ~15 hrs in 4 ml LB, I get ~500ng/ul (50 mls in all). So 25ug in all. Is that too much, too little...any sugestions?

[swanny]

Depends on whether your plasmid is high-copy or not, but 25ug from 4 ml of culture sounds fine!

[DNA]

swanny, on Oct 28 2009, 01:03 PM, said:

Depends on whether your plasmid is high-copy or not, but 25ug from 4 ml of culture sounds fine!

I got 11 uGg from 4 mL culture.Is that Ok for a low copy plasmidZ?

[pDNA]

Its all okay ...as long as it is the right plasmid or at least plasmid DNA.

So do not rely to much on those spectrometer readings ...put the DNA on an agarose gel an quantify it there (this way you are able to make some qualitativ statements about your preparation, e.g. is it supercoiled or degraded).

If you really done a miniprep (you stated that you get 50ml of 500ng/µl ...this is not a mini-prep ...thats more a terra-prep

So i think you meant 50µl

As far as i know 25µg is the amount a standard-column can bound ...so this is the maximum amount that you can elute from a column.
Very good prep results i think!

Regards,
p

[phage434]

The nominal capacity of a Qiagen column is 10 ug, so this is quite high, especially for a low copy number plasmid.  I would repeat the suggestion that you quantify by running a gel against a known concentration marker.