Would u plz give me some suggestions? - (Jun/02/2006 )
I agree that it sounds pretty weird abt he 1µg in 1hr, but I am working with this in mind atleast for mluI digests and it has been working fine. Its difficult to be very precise so thats y I wrote use 0.5µl or 5units for what the amount of DNA u used.
Invitrogen has MluI enzyme and I compared it with NEB and found it to be better. I use the enzyme from invitrogen and NEB buffer. Actually this is trhe first time i used an enzyme other than one from NEB.
Also I inactivate the enzyme at 65C for 20 min.
Again, this is my experience with this enzyme. I experiemtned with dif. amounts of enzyme from bot NEB and invitrogen and after a week, I got it right and am sticking to it.
The way I figured it was, I did single digests with Mlu1, different conditions (enzyme units and incubation time) and religated the vector. After transforming this, u can have an idea which condition suits u or is better.
Hi.
When I said 13kb and 10kb bands I assumed that use cut your vector with two enzymes (eco and mlu) to get a 10kb fragment and get rid of a 3 kb part of the vector? Am I right? Thats why if you cut with single enzuyme you will have whole 13kb linearized vector and otherwise 10kb! Clarify if my assumption about vector digestion is right.
Invitrogen has MluI enzyme and I compared it with NEB and found it to be better. I use the enzyme from invitrogen and NEB buffer. Actually this is trhe first time i used an enzyme other than one from NEB.
Also I inactivate the enzyme at 65C for 20 min.
Again, this is my experience with this enzyme. I experiemtned with dif. amounts of enzyme from bot NEB and invitrogen and after a week, I got it right and am sticking to it.
The way I figured it was, I did single digests with Mlu1, different conditions (enzyme units and incubation time) and religated the vector. After transforming this, u can have an idea which condition suits u or is better.
Thank you again, scolix,I think the NEB MluI is not so good due to the long transportation, thus when I use 1µl in 50 µl volume, 37 degree for 5.5 hours, it is only partial digested, last day I changed the time to 8.5h, and it is totally digested, maybe Invitrogen enzyme and MBI is better, but better means expensive, it is a paradox.anyway,so thanks
When I said 13kb and 10kb bands I assumed that use cut your vector with two enzymes (eco and mlu) to get a 10kb fragment and get rid of a 3 kb part of the vector? Am I right? Thats why if you cut with single enzuyme you will have whole 13kb linearized vector and otherwise 10kb! Clarify if my assumption about vector digestion is right.
Right, what you said is totally corret. and yesterday while I did the double digestion, meanwhile I did the single digestion with MluI, and after eletrophoresis, I saw a 13kb in the single, and 10kb,3kb in the double digestion, it is really nice, cause it is completely digested, and the place of 13kb is different from 10kb, I am so glad about that, thanks again