help. Is this double digest right? - (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!
You might be able to digest this way but I would use less enzyme and more volume. I don't know how much DNA you're digesting but if I were doing this, I would start with 0.5uL AscI in 10uL rxn, incubate 1hr at 37C then heat inactivate. Then add 0.5uL BglII, 3uL 10X Bfr 3, and bring your volume up to 30uL (I wouldn't bother purifying until I know that this method does not work well.) After running your rxn on a gel, you should be able to tell if it was successful. To be thorough, you can run controls of single enzyme digests and undigested.
As mentionned before it's far too concentrated the golden rule is
1 unit enzyme cuts 1 microgram of DNA in 1 hour
I always perform my digest in 20 microliters with 0.5 microliters of enzyme (usually 10u/microliters for most companies)
Judes' suggestion is a good idea. Don't bother cleaning up your DNA between reactions because you will lose too much and it is unnecessary. The most important ingredient in restriction enzyme buffers is NaCl. As long as the final NaCl concentration is correct your particular restriction enzyme it will cut well. In your case, AscI likes 0 mM NaCl (buffer 4) and BglII likes 100 mM NaCl (buffer 3). So you start off in buffer 4 with AscI as you've done and then add the salt (using buffer 3 or adding your own NaCl solution) when you want to digest with BglII. As long as the final concentrations of NaCl are at these levels (0 mM for AscI and 100 mM for BglII), your digests will work well.
You can go to the fermentas website. Thy have a tool called DoubleDigest.
It tells you in what conditions to do any digestion.