10:00 A – 8:00 P, 9:00 P – 11:00 P
Joint project
- Help Hao relocate cells from yesterday and check sequential hybe
- Succesfully relocated all cells.
- Substantial Cy3 signal still present, but much more Atto647N.
- Need to analyze images.
STORM analysis
- Still running round 1 ph-M ph cluster STORM analysis
OligoPaints
DNA clean-up
- take 1:10 dilution sample of all pooled PCR reactions.
- Column clean up of pooled PCR reactions. Spin in 50 mL flasks to collect flow-through that doesn’t bind in first round
- wash with 4 mL EtOH wash buffer on vacuum manifold.
- Quick spin in discard columns to elute extra EtOH buffer — this is critical.
- Elute fresh MilliQ ddH2O.
- Repeat whole procedure with original elute. (Potential issue: haven’t gained much concentrating effect, from 1 mL to 300 uL).
- Gel
PCR probe making
- Troubleshooting PCR: BB recommends small scale PCR with first to amplify emPCR product prior to PCR with adapters.
- Ran small scale PCR with just short primers (new program, 30 rounds amplification).
- used 3 uL of PCR product (in 60-100 ng uL by NanoDrop following CC-5 cleanup column) to seed new PCR (3 rounds low temp 7 rounds high temp).
- need to calc concentration to see probe was actually made substantially.
- Tomorrow: Run adapter PCR on column. Use product (most of it?) to seed new batch of large scale PCR with PPT-cntrl primer
Embryo in situs
- Cold washout probe in 2x SSCT
- check staining on Turnkey. Not anything as strong as control, but potential staining with nuclear background…
- signal very strong on STORM2. Should have washed more, background very high. Still looks very promising for a night of imaging.
