I need help with the steps in sequential digests please - (Nov/30/2006 )

Hello, I need to do a sequential digest. I just want to make sure if after my first digest do I need to gel purify to start the other digest reaction, or if I can purify the digest without running on a gel and then make the other digest reaction and gel purify the cutted insert.

And last thing is, after I purify the first digest (in about 6uL) do I use all of this for the next reaction right?

any help will be very appreciated

i don't gel purif after first one.
i use all for second digestion. Btw i resuspend or elute in the volume of water required for the next digestion.
For ex if i need 5µl buffer 0.5µl of BSA and 2µµl of enzyme, i resuspend in 42.5µl

Thank you Fred!!

When I have to do double digestions with enzymes that work in different Buffers and then, purify my band. Some times (when I have small amount of DNA) I prefer to do the first reaction in a small final volume, then add the 2d enzyme and add more H20 and the right buffer (and BSA if it’s the case) to a large final volume.

For example:
Digest DNA with PacI using B4 (NE BioLabs) and BSA to a final volume of 25 ul .
Then add NotI, B3 and BSA to a final volumen of 75-100 ul. Run and purify.

Doing in this way, you don’t lose DNA purifying the first digestion.
It always works for me.