9:00 am – 5:45 pm
Plan
- need to do fewer review experiments at once so we can do them more carefully
- should focus on the live imaging and Pc analysis
- need to get good QPCR
- need to set up deep-sequencing
- need to analyze internal domains
- need to do 2-color experiments (try L2 G05 G06).
Lab meeting
- Jiang practice talk (see notes)
RNAi QPCR
- started from RNA again, ran total RNA prep using oligoT primer
- spiked in 2 uL RNase H as per the SS3 kit, incubated at 37
- set up QPCR reactions
- diluted DNA 1:5, then added 2.5 uL per tube.
- revised quality controls on QPCR analysis script
- most genes no amplification
- Dfd amplified inall the Ph KD but none of the controls
- probably not enough cDNA (or maybe inhibitor?)
Meeting with XZ
- live imaging plans:
- move forward with analysis of SIM data
- focus on the TIRF2-telo
- ph
- internal domain analysis
- domain cross-boundary interaction
- qPCR
- sequencing
- immuno-staining (?)
- western (?)
- other things are lower priority.
TIRF2 imaging
- test imaging TIRF2-GFP fusion
- these look very encouraging
- clearly visible on Turnkey by eye
- lots of spots, low nuclear background, clear signal.
