The protocol for double digestion with BamHI and SacI - (Sep/22/2009 )

Sorry did you really mean 100 µg/µl? or did you mean 100 ng/µl? 100 µg/µl seems quite unrealistic to me...Lets say you have 100 ng/µl and you use 3.5 µl. that means you digest 350 ng. This is not enough to see the 100 bp fragment in my opinion. Try digesting up to 10 µg with 3 U BamHI per µg and then purify and then digest everything you get (!!) with 3 U SacI per µg DNA. see if this works. For the second digest, if you get 20 µl DNA, then perform the digest with all of it in a total volume of let say 30 µl and put all of it on the gel. You have to consider that lets say your palsmid is 3000 bp, then you have 350 ng. The 100 bp fragment therefore corresponds to only about 10 ng of your total 350 ng! that is not enough to see properly on the gel. If you digest 10 µg then to about 300 ng which you should be able to see and cut out the band.

Stardust

PS: could you attach a gel picture?

stardust on Sep 25 2009, 05:05 PM said:

I agree with stardust. I often digest 1ug vector containing about 200bp insert with 10U enzyme such as xhoI, xbaI, then run all the digestion reaction production on the gel. I can see the 200bp fragment clearly.

Hi there!

Did it work?

Stardust

Hi all!
I am also facing some problem using these two enzymes. I am trying to digest pQE30 vector with BamHI and SacI. When I digest about 500ng of my vector with BamHI (using ~6 units) in a 20ul reaction, even after 4 hours of digestion I am able to see the plasmid undigested on 0.8% agarose gel. The NEB catalog says that BamHI shows immediate activity, then why am I getting the uncut vector? (the enzyme is also new)
And I have been performing double digestion for quite a time but I am not getting a double digest. I have tried to digest sequentially as well as both enzymes together in the same reaction. But in all the cases I see the vector self-ligate upon ligation.

I need some help please!

I strongly suggest doing this reaction in a higher volume. Your DNA likely contains inhibitors to cutting, which will be diluted when you make the DNA a small fraction of your final reaction.