Restriction enzyme digestion - procedures in restriction enzyme digestion (Apr/01/2007 )
hello guys.. I'm new here. I would like to greet you guys once again... HELLOO 
I did an experiment on restriction enzyme digestion on pAMP and pKAN. However, there is a step that make me wonder.
It is that we have to place the tubes containing digested plasmid in 70oC water bath for 15 minutes as the final step after we mix them with enzymes, centrifuge, and incubate at 37oC.
Could anybody help me.. 
The actual restriction enzyme cutting occurs at 37C. The incubation at 70C does what is called a "heat kill" of the enzyme, which denatures the protein (enzyme). This is not strictly necessary in most cases, but is helpful if further processing of the DNA without purification is anticipated. The DNA will probably run on a gel and give the correct bands without the heat kill. You could try an experiment and let us know the result -- run some with and without the heat kill. What is the enzyme? Some enzymes might stick to the DNA and change the apparent fragment size.
thanks a lot phage 434. so kind of you. however, i dont think i can run the experiment again ( for the nearest time, but maybe soon i would ) 
. Im only a fresh student, the experiment is in my syllabus. But, someday when i have the opportunity, i will do it. The enzymes used are HindIII and BamHI to cut pKAN and pAMP plasmid. However, thanks a lot. I appreciate the knowledge u give to me.
I did an experiment on restriction enzyme digestion on pAMP and pKAN. However, there is a step that make me wonder.
It is that we have to place the tubes containing digested plasmid in 70oC water bath for 15 minutes as the final step after we mix them with enzymes, centrifuge, and incubate at 37oC.
Could anybody help me..
Hello hourgh,
I understand that you misunderstant the methods you wrote. you can use the methods below...I think you can achieve to do it.
for restriction digestion of plasmids
Reagent: ddH2O 18 µl, NEB buffer 2 (10x) 3 µl, BSA (10x) 3 µl, BamH I 1 µl and Hind III 1 µl ....totally 26 µl
Enzyme mix 26 µl, pAMP 4 µl or pKAN 4 µl..... total volume is 30 µl
Than, incubate the samples at 37°C for 1 or 2 hour to have restriction digestion occur.
After the didestion, place the samples at 70°C for 20 minutes to inactivate the restriction enzymes.
Do it and achieve. bye
fberis
I understand that you misunderstant the methods you wrote. you can use the methods below...I think you can achieve to do it.
for restriction digestion of plasmids
Reagent: ddH2O 18 µl, NEB buffer 2 (10x) 3 µl, BSA (10x) 3 µl, BamH I 1 µl and Hind III 1 µl ....totally 26 µl
Enzyme mix 26 µl, pAMP 4 µl or pKAN 4 µl..... total volume is 30 µl
Than, incubate the samples at 37°C for 1 or 2 hour to have restriction digestion occur.
After the didestion, place the samples at 70°C for 20 minutes to inactivate the restriction enzymes.
Do it and achieve. bye
fberis
is there any reason that you keep the reaction volume 30 ul???
i will follow as below
pAMP or PKAN 1 ul
BamHI 1 ul
HindIII 1 ul
Buffer (x10) 1 ul
H2O 6 ul
total 10 ul in one tube place in 37c for digestion and check by electrophoresis
1ul is a lot of enzyme for a 1 hour digestion. Enzymes are typically rated to cut 1ug of DNA per hour per unit of enzyme - and 1ul is usually 10 units. So 1ul can cut 1ug of DNA in 5 minutes.
Promega recently tested 30 enzymes and found this to be the case for 27 of them.
So, either save money and use 0.1ul of enzyme, or save time and use 1ul of enzyme for 5 minutes. (I do both depending on how much enzyme I have left in the tube 
)
)me too