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
It seems pointless to me to get rid of your DNA unitl sometime after your IP.............I would do the DNAse treatment after the reverse-crosslink step and before the ProK treatment. Usually most people pull either 1 or 10 percent of their input..................so if you have 300ul of material your about to IP I would pull 3 or 30ul and use that material in your %Input calculations.
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
its fine that your DNase and reverse crosslink steps are in one combined step. 10% Input would be, correct, out of the initial material. However, say you dilute your 300ul into 3mls of dilution buffer and then aliquot that into three 1ml IPs, then to take a 10% Input smaple would be to take 100ul of the diluted material before you aliquot it into 3 separate IPs...........10% Input is an awful lot in my opinion................I (and most people I know) only take a 1% Input sample.
thank you a lot!)))