making insert from miniprep DNA - (Feb/10/2007 )

hi all
do u have any experience to make insert from miniprep DNA??
if u are successful in ligation using insert making from miniprep DNA , could you please share me your experience with me???
thank you

Nearly all of the ligations that we do in our lab are from miniprep DNA. Only the final product goes to the maxi step.

We prepare DNA without using columns, using all the 3 buffers, spin 10 min, take supernatant and mix with isopropanol, spin again, wash with 70% ethanol and elute.

There is nothing so special about using insert from miniprep DNA. One only needs sufficient amounts of DNA to digest out the insert and thats all to it.

dear scolix
dont u use phenol:chloroform treatment in miniprep???
and how u calculate the DNA amount from mini prep sample???

we also done miniprep using the same way as you, but in my lab they think miniprep sample is too dirty to use in ligation.

what is your DNA yield from 2 ml over night culture???

thanks for your reply

I have never used phenol-chloroform treatment till now. I somehow get by without it.

The DNA amount from miniprep is a crude estimation. I have estimated there is more than 300-400 ng per ul in a 50ul elution from a 2ml ON culture.

The miniprep DNA is dirty for sequencing but not for digesting and using the insert for ligation. We do it routinely.

then how do u remove protein from miniprep DNA

these are really vrey useful info for me, thanks a lot

We dont remove anything. Its not the cleanest DNA but sufficient for digestion and further cloning.

I do phenol chloroform extratcion adn get better yield than using kits, but as scloix said, it is not as clean as using kit.
150 ng/ul in 50 ul with 3 solutions
100 ng/ ul in 33 ul with kit
using nanodrop

I think you do not need to use phenol-chlorophorm extraction if your cells have no endonucleases. In our lab, when we do not need a particuliarly clean DNA, we extract DNA with home made solutions, precipitate the DNA with isopropanol, wash once with 70% ethanol and solubilize in clean water. You sure have to digest with RNAse to get rid of the RNA, but you can get pretty good DNA.

Saves a lot of money and time (it's even faster than with the Qiagen kits)

my cells have EG's

my cells have EG's