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===May 30, 2013=== * I isolated 2 more cells this morning, using smaller pipettes, still using pull = 600 on the puller. ** Yesterday I used heat = 700. Today I used heat = 800. * I also made more careful dilutions today, since I already know the ballpark concentration from yesterday. ** Diluted 200 ul cells in tube with 800 ul PBS (1:5), then diluted 100 ul of this with 1000 ul of PBS plus 100 ul 0.5 M EDTA (1:12) --> 1:60 dilution total, with 0.042 M EDTA. ** Each 3 ul droplet of this solution contained ~30 cells --> 1e4 cells/ml is a good working concentration. [[File: 2013_05_29_lymphocyte3.tif|400px]] Lymphocyte #3 [[File: 2013_05_29_lymphocyte4before.tif|400px]] Image of the glass top tube before expelling lymphocyte #4. [[File: 2013_05_29_lymphocyte4.tif|400px]] Lymphocyte #4 * The quality of these images is obviously questionable. It is very difficult to determine the presence or absence of a single intact cell without any kind of stain or dye. One option is to use trypan blue, which should be excluded from live cells and therefore won't interfere with RT primer annealing. ** Jeff suspects that DAPI interferes with MDA, which is one of the reasons I have been avoiding any kind of staining. * At this point, I am strongly leaning towards FACS to sort single cells into a 96 well plate. ** If I could sort into RT primer annealing solution, then I wouldn't need to worry about excess volume. ** Blue told me that he has successfully sorted into 2 ul, so 1.2 ul isn't too far off the mark. ** 96 well is probably preferable to 384, because it is more likely that the cell with hit the bottom of the plate rather than stick to the side somewhere. * I need to design a primer to check the library quality before sequencing. I can still used the same gene specific primers I designed for the pre-IVT check, but I need a new forward primer. ** I can used the 3' adapter region.
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