Posted 15 February 2012 - 04:55 AM
I've recently started working in this new field of animal tissues and mammalian cell lines (originally I am molecular microbiologist).
Well I am working with lncRNAs now, at the moment trying to validate, which lncRNAs bind to histone H3 in ES cells, in murine hearts and muscles.
Since I am doing RIP, not ChIP, there are certain differences, of course. I am using Millipore MagnaRIP kit, combined and adjusted with tons of other protocols (including Active Motif and those, sent to me by our collaborators). So in fact i need to get rid of DNA after lysing samples, but before incubating samples with beads-Ab complex. And I have no clue where I should stick this DNase treatment step (as far as i understand DNA will actually disturb proper binding of my histone H3-RNA complex ), in which proportions I should add enzyme and reaction buffer.
Please help me!!!
and one more stupid question. amount of input should be proportional to total amount of lysate?? say I get 300 microliters of lysate supernatant, how much I should keep as input???
Please don't laugh at me, I am actually not that stupid.. It's just a totally new field for me and I've just started...
Posted 23 February 2012 - 01:25 PM
Posted 28 March 2012 - 08:15 AM
Thank you so much for your reply!!
But the thing is that decrosslinking and PK treatment are in 1 step in my protocol...
and 1 more question - 10% input out of 300 ul of material.. of total material??? or from an aliquot I use for each IP? I thought so....
Posted 28 March 2012 - 08:28 AM
Posted 30 March 2012 - 12:49 AM
thank you a lot!)))