8:45a – 4:50p, 8:45p – 11:45p
Goals
- re-read review — complete
- finish discussion — complete
- Stop IVT reaction, start RT reactions. Freeze RNA. — complete.
- talk to Hazen about fixing Hal. — fixed.
- call Calgary rental car. — Didn’t do.
- call Beckmann sales office: (800) 526-3821. Beckman-Coulter A63987 1 40 mL $684 Agencourt RNAClean XP magnetic bead. — Ordered.
for cluster analysis:
- use beads to warp images instead of auto-correlation
- implement dual thresholds for identification of ‘real clusters’, and separate threshold for ‘dot found on this day’.
RT reactions
- G2 + G3 purified: 15 uL RNA + 5 uL primer + 2uL 10mM dNTPs + 2 uL ddH2O in 40 uL reactions
- G2 + G3 purified no primer: 5 uL RNA + NO PRIMER + 1uL 10mM dNTPs + 7 uL ddH2O in 20 uL reactions
- G2 + G3 raw, 3 uL RNA + 5 uL primer + 2uL 10mM dNTPs + 14 uL ddH2O in 40 uL reactions
- per 40 uL reaction: 8uL 5x buffer + 2uL RNasin + 4 uL .1M DTT + 2 uL SS3
- per 20 uL reaction: 4uL 5x buffer + 1uL RNasin + 2 uL .1M DTT + 1 uL SS3
- at 50C for 2 hours.
- Degraded RNA via alkaline hydrolysis at 95C.
- Oligo-cleanup, eluted in 16uL ddH2O. Spec tomorrow. In small fidge now.
STORM
- STORM imaging of G2 with 561 beads (1:10,000).
- beads substantially too dilute — many cell fields have no in-focus beads. Try 1:2,500.