Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Digestion of mini-prep by mistake - (Mar/04/2011 )

Hi, everyone
Recently I just isolated some of my clones by mini-prep.
I was going to test the construct by double digestion.
I planned to take 5uL of the construct for digestion.
Unfortunately, I added the enzymes and buffer into the whole construct.
Can this be saved?

Many thanks

-Keane-

You probably have incomplete digestion, so you can likely transform the digest into cells to recover the plasmid (assuming you don't have the original cell stock, which you should have saved in any case).

-phage434-

i'm afraid not!
...but you can try to do isopropanol precipitation and resuspend the pellet in 10ÁL H20 and electroporat that ...maybe you will get colonies.
But in general i would re-start from the colonies ...because it will take you the same time for the re-purification of your plasmid.

Regards,
p

Keane on Fri Mar 4 15:14:21 2011 said:


Hi, everyone
Recently I just isolated some of my clones by mini-prep.
I was going to test the construct by double digestion.
I planned to take 5uL of the construct for digestion.
Unfortunately, I added the enzymes and buffer into the whole construct.
Can this be saved?

Many thanks

-pDNA-

I agree with phage. You probably have enough uncut plasmid to efficiently transform a few cells. Also, WHERE ARE YOUR GLYCEROL STOCKS?! Haha! I know the lab across the hall from mine makes glycerol stocks for EEEEVERYTHING!

-LabLackey-