12:30 pm – 11:30 pm
(HHS appointment in morning for crushed foot).
Making RNA probes through old method as a control for RNA staining quality
- found sna and hb probe templates
- hb tube is empty, attempted to dissolve DNA from evaporated sample with added H2O (10 uL).
- ran PCR reactions with m13F + m13R primers using Phusion
- 30 cycles (reset annealing temp?)
- run test gel (0.7% in TAE with gene ruler express)
- sna amplified, hb didn’t. Probably ran a bit too many cycles / not enough primer: some snail concatamers. Shouldn’t really cause problems.
in vitro transcription reaction (old style as control)
- following old protocol
- Per 20 uL reaction
- 4 uL 5x buffer
- 2 uL 10x dig-mix
- 1 uL T7
- 1 uL RNasin
- 1 uL DTT
- 5 uL DNA template
- 6 uL ddH2O
- Run as a 40 uL reaction
- 8 uL 5x buffer
- 5 uL 10x dig-mix (expired)
- 3 uL T7 (expired)
- 2 uL RNAsin
- 2 uL DTT
- 10 uL DNA template
- 10 uL ddH2O
- ran carb treatment, STOP treatment, and percipitate with LiCl overnight at -20C.
Manuscript edits
- discuss shortening with XZ
- revise text to shorten
- update calculator
- revise figures