Decide on concentrations of protein and RNA

Activity assay (RNA>>Kd) - DM15 + m7GpppRPS6 (Kd is around 20nM, so will use 1uM RNA to be in excess)

* Don’t prepare protein dilutions until later – after mixing other components *

Example calculation: For final [DM15] = 4uM, 5X [DM15] = 20uM

Kd (Protein>>RNA) – DM15 + RPS6 20-mer (Kd is ~776nM, 500nM corrected RPS6 20-mer)

Prepare 5X Binding Buffer – Refer to Lahr et al. 2015 NAR – Aliquot and store in -20C in Eppendorf tubes. Make sure to filter!!!

Pour native 0.5X TBE – Do this on the day of, or the night before

Check what percentage gel we want to run. Refer to chart at radiation bench. Want to know where our band will fall relative to the two dyes in formamide (Bromophenol Blue and Xylene Cyanol).
1. This time, I’ll be using a 7% gel for capped RPS6 20-mer. 10mL makes 1 gel
Clean plates – Alconox, EtOH, Acetone, EtOH
Pour gel. Determine % gel you need. Refer to very worn, taped graphs above rad waste. Example calculation is for making a 7% gel. Allow to polymerize. Roughly 10mL per gel.

7% PAC, 0.5X TBE

40% 29:1 Acrylamide (PAC)->	1.75mL
1X TBE---------------------------->	5mL
ddH2O---------------------------->	3.14mL
10% APS-------------------------->	100uL
TEMED---------------------------->	10uL
TOTAL VOLUME	10mL

General workflow:

Prep RNA (~30m) -> add nonspecific competitors, aliquot mastermix -> add protein dilutions to mastermix aliquots -> allow binding reaction to reach equilibrium (~30m) -> run gel (40-50m) -> dry gel (30m) -> expose O/N

Prepare binding reaction without protein

BINDING REACTION

2uL	Binding buffer (5X)
2uL	Protein (5X)
2uL	RNA (500 counts per reaction)
0.5uL	tRNA (1mg/mL)
0.5uL	BSA (2mg/mL)
3uL	ddH2O
10uL	TOTAL VOLUME

*** I end up making mastermix to use that contains everything but the protein because you’re titrating in protein. This means I calculate how much of everything I will need in the end enough for the number of reaction + 0.5 reaction, in case there’s pipetting error. **

Prepare 5X RNA. For Kd experiment, you want RNA<<Kd, so trace amounts of hot RNA. This means treat hot RNA as negligible. I do 500-1000 counts per reaction.
1. Snap cool to allow for proper folding.
  1. Water + RNA + 5X Binding Buffer
  2. 1m 95C
  3. 20-30m on ice
While RNA is on ice, prepare protein dilutions.
After incubation, add tRNA and BSA to make a “mastermix” that will be aliquoted.

Running native 0.5X TBE

Pre-run gel at 120V for 30m in cold room. You can also bury the apparatus in ice on rad bench, if there’s space.
Add 0.5uL loading dye to 0 protein sample.
Flush out lanes with syringe and needle.
Load gel and run for ~45m-52 in cold room or in ice. This depends on where you expect your RNA to run based on number of nucleotides.

Dry gel

Check to make sure that tubing is connected.
Tip gel onto filter paper – don’t try to remove the edges.
Turn on the dryer to heat the lid.
Place saran wrap over gel+filter paper.
Place saran wrap+gel+filter paper in between mat and plastic/rubber thing.
Turn on the vacuum.
Check to see that a vacuum was created, and allow to dry for 30m.

Expose

Blank a phosphor screen over light box for ~5m.
Place dried gel in cassette so that gel is face up.
Place white part of the screen over the gel. This allows the screen/plate to be exposed to radiation
Close the cassette, label with name, and let expose overnight.

Image using Typhoon – code is 1-3-5-2-4

Take phosphor screen and stick magnetic end to the plate.
Place cable ties into position.
Open the software.
Image, and save onto flashdrive.
Do not remove screen or open door until return button pops up, and you close out.