6 replies to this topic

[Curtis]

my mini-prep kit ran out of filter-columns and now I am extracting plasmid with conventional method (centrifuge based, solution I, II, III)

In the final step of conventional method we add water with RNase to remove all RNA. But I'm not sure if I can use these plasmids directly for transfection as long as the solution got RNase. I'm afraid it might degrade all RNA of host cells and knock out everything!!

[mdfenko]

how would the rnase enter the cell?

there are already rnases present in the cell. rna is made by the cell as necessary.

in other words, don't worry about it.

Edited by mdfenko, 25 October 2010 - 07:04 AM.

[Curtis]

thanks mdfenko,

I do know cells got RNse inside, but this is excess RNase we are adding over the cells, so I was concerned about it. Thanks again anyway...

[hematopoietry]

Curtis, on 25 October 2010 - 11:56 PM, said:

thanks mdfenko,

I do know cells got RNse inside, but this is excess RNase we are adding over the cells, so I was concerned about it. Thanks again anyway...

In some kits, the RNAse is already added in the resuspension buffer (which is why you keep these in the fridge). If you are worried about this, you could do this too for you miniprep.

[Curtis]

Exactly!!! in Invitorogen's PureLink kit we add RNase in the first step (in the resuspension buffer). There must have been a reason they thought of adding it in the beginning rather than ending. So perhaps I can do the same and add RNase to Solution I

[perneseblue]

The protocol I use for conventional alkaline lysis (Sambrook) on mini-preps, adds 100ug/ml of RNAse A  to Solution I.

[Curtis]

perneseblue, on 29 October 2010 - 08:14 PM, said:

The protocol I use for conventional alkaline lysis (Sambrook) on mini-preps, adds 100ug/ml of RNAse A  to Solution I.

yesss, thanksss,problem solved
that's a lot though because I have removed RNA even with 3ug/ml RNAse in the final elution step