Question

In old Alkaline Lysis, mini-prep procedure, RNase A was not added with the Alkaline Lysis solution (I or one). It is added with the TE buffer at the end of the purification procedure. A newer method of the modified alkaline lysis (Plasmid mini-prep kit) RNase A is added with the PD 1 (the first solution, the resuspension solution) buffer at the first re-suspension. What is the reason for adding RNase A in a different step of modified alkaline lysis mini-prep procedure when compared to the old alkaline lysis mini-prep?

Answer 1

Main reason is that in alkaline lysis protocol RNase was added in last step as high NaOH was added during intital stage in that stage if we would add RNase then RNase will get denatured at such a high alkaline or high pH so it's of no use. Once the RNA was purified in last step we add RNAse as at time buffer pH was 7.5

While in new Mini pre method, we add RNase in 1st step itself as in 1st we do lysis of bacterial cell using lysozyme which break down cell wall and so RNA along with genomic DNA and plamsid present in supernatant and this is the time at which RNA need to be degraded other wise it will also bind to silica gel column which attached negative charged nucleic acid including RNA.

Hope it's clear..thanks