9:00a – 12:35a
Goals
- work on Presentation for Stowers
STORM
- Ran out of disk space, crashed O/N
- Run lost focus anyway immediately after position 0. Probably not enough oil. Possibly positions were chosen too far apart. Need to explain these things to Bogdan to reduce future failed imaging.
- Setting up F1 run again with Bogdan.
Advising
- discussing rotation goals and lab organization for rotation
- characterize relation between chromatin position in nucleus and chromatin structure
- chromatin type and chromatin structure properties
- boundaries
- Helping Hao troubleshoot matlab code. Small bug in earlier matlab code shifting beads but not data by xshift/yshift correction.
- Team meeting and individual meeting tomorrow.
Probe Making
- IVT reactions finished
- Cy3 primers for lib2 arrived (primer 2 and primer 4)
- New samples: D11, D12, E1, E8, E10, E12, F1, F3, F4, F5
- Previously purified RNA: G3, G4, G5, AbdA1, Ubx1, (previously made and tested with commonP1 no 405).
- 15 reactions to remake previously made/ tested probes with P1-A405, P2cy3, P4cy4
- 10 reactions to test new probes with cy3 (better for quantification)
Protocol
- For each sub-library, mix the following in a single PCR tube or well of a PCR strip or plate
- 15 uL RNA template
- 5 uL 100 uM labeled primer
- 6 uL 10 mM dNTP mix
- 16x P2cy3-Master = 80 uL 100 uM P2Cy3-primer, 96 uL dNTPs, Aliquot 11 uL per tube.
- 6x 405-Master = 30 uL 100 uM 405-primer, 36 uL dNTPs
- 6x P4cy3-Master = 24 uL 100 uM P4Cy3-primer, 36 uL dNTPs
- Incubate at 65C for 5 minutes, then mix in the following
- 8 uL 5X First-Strand Buffer
- 4 uL 0.1 M DTT
- 1 uL Murine RNase Inhibitor
- 1 uL SuperScript III
- 28x Master = 224 uL buffer, 112 uL DTT, 28 uL RNase Inhibitor 28 uL SuperScript III
- Mix via pipetting and incubate at 48C for 3 hours.
Gels
- Make new
- 12g Urea
- 7 mL 40% acrylamide (19:1) Bis
- 2.5 mL 10x TBE
- 5.5 mL ddH2O
- 38 uL 10% APS (ammonium-persulfate). Make fresh
- 38 uL TEMED
- need to let cool before adding APS and TEMED!!
RT results
- Promega buffers failed completely to work with NEB T7 or interfered with RT. Test RNA clean up or test gel tomorrow
- old premixed T7 worked poorly — these samples also had critically low template concentrations.
- Fred’s library version of Ubx and AbdA don’t have my Lib2 common sequence, can’t use new primers silly!
