Sequential digestion protocol - (Oct/22/2008 )

Can anyone suggest me a protocol for sequential digestion of vector pet16b with BamHI and NdeI?

I tried double digestion with no success, I also tried sequential digestion (NdeI first) but still with no success!Since I cant see if the second enzyme cut the vector I only can assume it, right??and something else, after the first digestion(and purification) i end up with a 30-50ul DNA in water. My question is how am I suppose to use this for the second digestion?Use all of it and make a total reaction volume of 50ul or what?

plz help...

I'll do a sequential digest for this one, starting with NdeI (as you have done). The NdeI and BamHI sites are too close together in this plasmid. Do the digest in NEB buffer 2.

Do you mean 30ug-50ug? or 30-50ng DNA in water?

Well the first thing is to start off with enough DNA. Typically I would digest 10-25ug of DNA. Yes, it is alot and people would say it is extremely wasteful. But you grow your own plasmid, and you don't ever want to face problems of not having not enough plasmid to work with.

Once the NdeI digest in Buffer 2 is completed. See if the plasmid has been linearised on an agarose gel. If the plasmid is linearised add BamHI emzyme. BamHI is a robust enzyme and will work in any NEB buffer. 1,2,3 and 4. You don't need to gel purify between steps. And yes, you can only assume that the BamHI has done its work. You cannot easily determine if has.

My digestion mix is as follows
x ul DNA (25ug)
10ul NEB buffer 2
5ul BSA 100x
1ul NdeI
total volume 100ul. Digest overnight. (I find it easier this way. Go home and comeback the next day and it is digested)

Add 2ul BamHI. Leave 5 hours. The DNA should be digested. Gel purify.

I mean 30μl(or 30λ or 30 ul), its volume.
many thanks... I'll try it...