I am planning to have endonuclease Ⅳ and Bst/Taq work in the same buffer, but definitely in different Temperature, i.e, the system will be passing first 37℃ then 60/95℃.
Can I simply put endonuclease Ⅳ in the isothermal-amplification/Taq buffer? Or if a new buffer is required, how should I prepare it?
- 1X Standard Taq Reaction Buffer Pack 10 mM Tris-HCl ; 50 mM KCl ; 1.5 mM MgCl2 ; (pH 8.3 @ 25°C) ;
- 1X ISOthermo amplification Buffer Components
20 mM Tris-HCl ; 10 mM (NH4)2SO4 ; 50 mM KCl ; 2 mM MgSO4 ; 0.1% Tween® 20 ; pH 8.8@25°C
- 1X Endonuclease iv Buffer 100 mM NaCl ; 50 mM Tris-HCl ; 10 mM MgCl2 ; 1 mM DTT ; (pH 7.9 @ 25°C) ;
Dear Zonghan,
in the end, you need to check by experiment (might make sense esp. if you have a lot of samples to process), but the Endo IV buffer has a significantly different pH and about double the salt, compared to the ISOthermo buffer. Maybe adding NaCl, Magnesium chloride and Tris-HCl can do the trick, if you're lucky.
Instead, you could use a PCR cleanup kit to remove the buffer components or possibly do a buffer exchange by gel filtration with Sephadex G 25, there are ready to use spin columns available.
What is the goal of your assay? I do have the same concern of the answer above, pH 7.9-8.8, 50 mM KCl ; 1.5 mM MgCl2 vs 10mM MgCl2. I a not familiar with the Mg differences, but you could try if you have lots of samples and assess which enz works or not.
I would instead use high fidelity PCR or PCR clean up kits that may save your time.
You must try and standardize in case you have large amounts of sample.
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