Help, sequestial digest - (May/21/2008 )
Hi, It is my first time to perform sequestial digest (Bgl II-Asc I). I don't know it is right or not. First, i used 1 ul AscI, 3ulDNA, 1ul Buffer 4, 5ul H2O, 37C,1.5h(Total Volume 10 ul). Then I directly added 1.5 ul Buffer 3, 2ul H2O and 1.5 ul Bgl II for 1.5h, 37C(Total volume 15ul). So can you tell me whether it is right? Thanks a lot.
What you did was incorrect :-(
I'm assuming you are using NEB enzymes.
What you need to do is digest your DNA with AscI in NEBuffer 4 first. You would want to have a total volume of about 20uL.
After you complete your hour long digestion, you should first heat inactivate the enzyme for 20 minutes at 65 degreees. After heat inactivation, you should then clean up the reaction by running it through a column. I recommend the Qiagen Quick spin PCR clean-up columns. This will remove all traces of buffer leaving you with pure DNA that has been AscI digested.
Elute your AscI digested DNA from the column using 30uL of elution buffer from the kit.
Next, add 4uL NEBuffer 3, 4.5uL water, and 1.5uL BglII. Incubate that at 37 degrees for an hour or so, and then gel purify the DNA, or run it through another spin column!
Make sure you have enough starting DNA. Each time you purify DNA through a spin column, you lose some. I always account for a 20% loss for each purification. Remember that if you need to have a LOT of starting DNA, you can always EtOH precipitate your DNA to bring down the volume!
I hope this helps!