Etchevers:Notebook/STRA6 in eye development/2009/05/04
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Sadaf C7 2nd week
New C7 immunoprecipitation, using last week's prepared chromatin (500 μL left). Sadaf is doing all the gestures herself. 125 μL x 2 C7 using 50 μL divided by 4, washed beads as per current protocol.
Long group meeting this afternoon.
Need to type up protocol in English now.
Also settled on six amplicons that we would expect to be enriched in the C7 and possibly the H3K4me3 fraction, but not the IgG ChIP, whereas there are two amplicons we would hope not enriched in any of them.
The two that should not be enriched are: ENm013 and ENr123.
For both C7 and H3K4me3: ENr324, ENr334, PGCP, GDF6, possibly Desert1 but it's borderline, FOXG1 only H3 signal in K562 cells.
For only C7: Desert1, FOXG1 only H3 signal in K562 cells - they both could go either way (+/- H3K4me3).
We've set aside ENr231, DLX5, CCNE2, Chr8 ctrl, Desert 2, PTDSS1 and SDC2 for efficiencies that are between 1.724 and 1.85, and some have poor Tm curves as well.
We have so far carried out four qPCRs: Sadaf ChIP 29-4, 30-4 and 1 mai #1 and #2. The last two are checking serial dilutions of the second series of primers, and include two primers of Stan's for CDH16 and an unspecific sequence that had been inefficient - and they still are. The first is also an efficiency calculation for the first set of primers, and the second is a actual trial for some of these on the first ChIP reaction (second ones will be tested tomorrow).
While I'm here, I counted only 300K cells in a confluent T25 for R1113 and froze 1/2 of these, reseeding 1/2 in a collagened T25 that is a bit old (PBS and kept in incubator). But I think there are more cells. There is an earlier aliquot of R1113 also in the liquid nitrogen. 64 places in the 5th row of the yellowhandled rack, these are in places 1 and 2.