Making blunt and stick end vector - (May/04/2006 )

Hello All,

I want to make SphI blunt end and BamHI sticky end vector. The method I am planning to follow is as follows:

1. 1ug of vector in 30ul digestion mix using SphI for cutting SphI site. Buffer I will be using here has following concentrations - 33mM Tris-acetate (pH 7.9), 10mM Mg-acetate, 66mM K-acetate and 0.1mg/ml of BSA)
2. Digest for 2 hours at 37C
3. Heat inactivate the restriction enzyme to continue for fill-in reaction using Klenow fragment & dNTPs. I will only add 5 units of Klenow fragment in the above mixture and continue further)
4. Perform fill-in reaction for 10 mins and then heat inactivate again and run it on the gel
5. Gel purify blunt end vector.
6. Continue with BamHI digestion to make sticky end
7. Digest for 2 hours
8. Gel purify the blunt and sticky end vector.

I am NOT planning to use Phosphatase during this digestion steps, just to make blunt ends work well.

The issues involved here are
1. I am gel purifying twice so I am expecting considerable loss of vector during 'cleaning up'
2. The Klenow fragment buffer is 500mM Tris-HCl (pH 8.0), 50mM MgCl2 and 10mM DTT) I will be substituting this buffer using the same buffer I am using in restriction digest) The company that we buy enzymes from claims that Klenow fragment would work 75-100 in the restriction buffer that I will be using.

Questions:
1. Since I would be losing a lot of vector in gel extraction, how much of initial vector, I should start off with for digestion? Is 1ug enough to have at least 200ng of digested vector to continue with ligation?
2. How do you find out, how much of DNA 1 unit of restriction enzyme would cut? I am not talking in terms of definition which says cutting of HindIII site in plasmid ABC and blah blah blah. If there is any simple definition...
3. Tris and Mg concetrations are high in Klenow fragment buffer system, anybody knows why?
4. What is the role of DTT in Klenow fragment buffer system?

Many thanks

Anybody for help here please

1. For such a procedure, i usually try to get 10ug of DNA and proceed from there.

2. Approx. 1 unit is needed to digest 1ug of DNA in 1hr. But a lot depends on the overdigestion quality of each enzyme. eg. After digestsing with BamH1 , one can religate 95% of DNA after 20 fold over digestion. This means there is some amount fo freedom for the time you could leave the digest. But if its only 10 fold, then I will try to keep the digestion time as low as possible.

I donot know abt the other 2 questions.