User:Ferdinando Pucci/Notebook/TEMs profile/2008/04/28

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TaqMan PreAmp

  • cDNA from 4 sortings, Mono6-9, CD115+Gr1+ inflammatory monocytes and CD115+Gr1- resident monocytes, 12.5ul each
  • 28 assay pooled: Angpt1, Angpt2, B2m, CD163, SDF1, EDG1, bFGF, VEGFR1, IGF1, VEGFR2, MafB, RelB, MMP2,9,12, Tie1, Tie2, LYVE1, EGF, Id3, Bv8, CXCR4, CX3CR1, CD62L, Arg1, YM1, CD115, GAPDH. 1:100
  • cDNA done with Oligo-dT and poliA as carrier

N202 injection

5*106 cells/mouse, 50 mice and 4 PGK-GFP. Kept 3 15-cm plates in line.

TaqMan PreAmp plates

Plate Genes
#1 Bv8, Id3, EGF, Tie1
#2 MMP12, CXCR4, MMP9, CX3CR1
#3 YM1, CD115, Arg1, Tie2
#4 GAPDH, Angpt1, Angpt2, MMP2
#5 SDF, CD62L, VEGFR2, B2m
#6 VEGFR1, bFGF, RelB, MafB

IFN exp3

RNA extraction from organs (Bra, Lun, Hea, Liv, Gut, Spl, Kid, BM) of mice BMT without tumors, long term. DNA+RNA from BM:

  • RNeasy mini plus kit
  • Lysate without EtOH on column, 30 max rpm
  • EtOH 70%, 1:1, RNA as the others (with DNase treatment)
  • AW1 500ul
  • AW2 500ul 2' 11k rpm, change tube
  • 1' 13k rpm

EB1 (Tris-HCl) 70 ul, 1' RT, 1' 13k rpm