9:30 am – 6:20 pm
- finalizing presentation of comparison of pipeline5 and pipeline4
- L8 comparison failed to save before I excited to allow JM sufficient RAM to run. Should have checked first!
- primers shipped today, should be here.
- set up PCR reactions
- Phusion calculator for new primers recommends 64C annealing
- 94C melt 3 min start
- 94C 30s melt, 62 C anneal, 72C extend, 34 cycles
- 72 elongate 10 min, 4C hold
- primers, diluted to 100 uM, run at 500 nM (1:20 dilution, 2.5 uL in a 50 uL reaction).
- 1:20 dilution of yw genomic DNA. Use 1 uL
- current dilution of Kc DNA – lets spec
- 2x Phusion HF master mix.
- sample order: Kc-DNA: Th, Rho, Pc-v2, Psc, YW-DNA: Th, Rho, Pc-v2, Psc
- row order: (Kc-DNA): Su(z)12, SCE, Ph-p, Ph-D, (YW-DNA): ESC, Su(Z)2, SCM, Pho
- on treatment of Su(z)^1b.8 cell line from Schwartz lab
- “We grow them on Schneider’s media from Lonza + 10% FBS. You need to use trypsin to passage the cells, much the same way as it is done for mammalian adherent cultures. And do not dilute the cultures more than to 20% confluence.”
- registered for SDB meeting (on Pcard)
- MERFISH lunch team meeting
- discussion with Sharmistha (RK lab) on image processing