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