I am isolating protein from more volume of culture. However I am unaware how much dnase to use and also what buffer to use for its activity, if I am dissolving the pellet in 30ml of lysis buffer which does not containing MgCl2 and CaCl2.
Try this (working at 0°C –water/ice; constant agitation):
Dissolve the pellet with Tris-HCl 50 mM pH 7.5, sacarosa 10 % (p/v) at ~10 ml/g.
Add the same volume of Tris-HCl 50 mM pH 7.5, NaCl 200 mM, glicerol 5 % (v/v), DTT 1 mM y PMSF 1 mM.
Add 300 μg lysozime/g pellet and incubate for 90min
Add MgCl2 (up to a final cc of 10 mM) and then add DNAsa I (up to a final cc of 10 μg/ml) Incubate for 30 min.
Try this (working at 0°C –water/ice; constant agitation):
Dissolve the pellet with Tris-HCl 50 mM pH 7.5, sacarosa 10 % (p/v) at ~10 ml/g.
Add the same volume of Tris-HCl 50 mM pH 7.5, NaCl 200 mM, glicerol 5 % (v/v), DTT 1 mM y PMSF 1 mM.
Add 300 μg lysozime/g pellet and incubate for 90min
Add MgCl2 (up to a final cc of 10 mM) and then add DNAsa I (up to a final cc of 10 μg/ml) Incubate for 30 min.
Hi,
I agree with Lucrecia - we also use 10 µg/ml DNaseI (final concentration). We occasionally do not add MgCl2 (as it disturbs some downstream applications), but it still works (probably there are enough Mg2+ ions released from the cells). You can use all kinds of buffers (Tris, HEPES, Phosphate) - choose the one that suites best for your purification procedure - the DNaseI is not affected so much by the buffer.
Good luck,
Magdalena
Also, might depend on your need, but a DNase is strictly not necessary for a protein purification since DNA is sheared off during lysis due to sonication.
Hi,
The User Guide for DNAse1 from Thermo-Fisher states that inhibitors include reducing agents such as DTT. All of the instructions are clearly outlined. Check out the manual!
Best,
Amanda
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