Rui:LabNotes/Collaborations/2011-7-12

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Prepare RNAseq library for sequencing[edit]

Size selection[edit]

Beads purification[edit]

  • 0.7X beads (35ul) to 50ul PCR, elute with 15ul
  • Transfer 12ul to new tube for sequencing, 1ul left for gel, 1ul left for Qubit

TBE PAGE File:7.12.11 final check.jpg

Qubit concentration

Sample ng/ul total ng
PC1 13.2 158.4
PC2 11.8 141.6
GFP+ 7.67 92.04
GFP- 11 132
E9.5 6.99 83.88
E11.5 11.4 136.8
E13.5m 12.4 148.8
E13.5f 10.5 126

Gel purification[edit]

Samples gel intensity relative intensity [conc.] V for 100ng Qubit [conc.]
GFP+ 221464.69 120725.2 40.77 2.55 7.67
GFP- 257057.98 152022.4 51.97 2 11
E9.5 207727.09 102391.96 34.21 3.04 6.99
E11.5 233881.54 128290.35 43.48 2.39 11.4
E13.5m 245476.91 132162.7 44.86 2.32 12.4
E13.5f 219923.48 120021.7 40.52 2.57 10.5
  • Qubit measurement is ~4 fold less than gel estimation
  • Pooled samples based on gel estimation, 14.86ul total with 14.86ul loading dye, divide into 2 lanes
  • Size of library is restricted b/t 200bp to 700bp, cut this region and purify DNA from gel
  • 400ul 1X TE, 37C shaking for 1hr
  • EtOH precipitation with 1/10V NaOAc and 2.5V EtOH, -80C 1hr, spin 30min, wash with 80% EtOH, airdry, resuspend in 10ul EB (total 20ul)
  • 1ul for gel check

File:7.12.11 pooled RNA libraris.jpg