Change in DNA size btwn mini and maxi prep - (Jul/18/2005 )

Hi,

I am having problems with my maxiprep.

My general protocol when making a new clone is to first do a miniprep, digest it with one or two enzymes to check that the size is right, sequence it and if everything seems right to do a maxiprep from the glycerol stock of the miniprep culture.

This time, there is a difference in size between the DNA from the miniprep and the DNA from the maxiprep. The miniprep DNA is approx. 6 kbp (which is right), but my gel shows that the maxiprep DNA is approx 12 kbp.

Is the DNA forming some sort of dimer? How can this be?

Any ideas?

Roshni

p.s. there is no PCR involved, I am just subcloning.

-Roshni29-

Are you RE digesting your maxiprep DNA to do the sizing?

-Daniel Tillett-

was is the quantity of DNA you loaded on your gels? sometimes, high quantity disturbs migration....

-fred_33-

QUOTE (fred_33 @ Jul 19 2005, 07:10 AM)

was is the quantity of DNA you loaded on your gels? sometimes, high quantity disturbs migration....

I digested 200 ng with EcoRI (to linearize the plasmid) and ran it on the gel alongside the undigested supercoiled plasmid.

-Roshni29-

turns out it my DNA ladder was degraded and running faster than it should.
it wasn't my constructs...phew!

thanks!

Roshni

-Roshni29-