Cell-free reactions

4 September 2006 – John Welsh

Pre-mix prep:

Thaw reagents on ice.
Calculate amounts needed for reactions.
- See <cytomim-ntp> and <PANox> under <cell-free reactions> folder.
Prepare pre-mix volume in slight excess of total number needed.
Pipette in order that reagents are listed on spreadsheets.
- Vortex after addition of C14 l.eucine.
- Pipette up and down to mix samples after this point.
- Discard tips in radiolabelled waste after this point.

Dilute plasmid as necessary – 1mg/ml for PanOx and Cytomim; 0.0667mg/ml for linear templates
Add volumes in Eppendorf or 96-well plate.
- Load 96-well plates in crease to enhance mixing.
- Add extract last; either pre-mix or plasmid can be first.
- Pipette up and down 10-20 times to promote mixing.
Incubate at 37°C – 3 hours for PanOx, 5-6 hours for Cytomim and linear templates.

Cut 3 pieces of filter paper for each reaction.
Label as T (total counts), P (total protein), and S (soluble protein)
Spot less than 1/3 of total reaction volume (4μl for 15μl reactions) on each piece of filter paper labeled T or P.
Spin down remaining samples for 15 minutes at 14,000rpm.
- If using plate, transfer to Eppendorf tubes before spin.
Spot same volume of supernatant on filter paper labeled S.
Place all samples under lamp for about an hour.
- Can dry overnight if necessary.

Place filter paper labeled P and S on 1L container.
Add enough 5% TCA solution to submerge samples.
- Keep on ice 20 minutes before pouring off.
- Repeat this step two more times.
Rinse samples briefly with water.
- (Preferred) Place samples under lamp for about an hour.

Place samples in vials for scintillation counter removing pins.
Make note of positions and numbering and rack numbers.
- T, P, S is a logical arrangement for samples.
Pump 5ml of scintillation fluid into each bottle, careful not to spill any into the rack.
- If any spills into the rack, remove vials and clean thoroughly with water and allow to dry.
Put caps on vials.
Label the top of each vial that starts a new row.
Put user plates 3, 5, or 6 on the first rack. (Each uses a 5 minute counting time.)
Slide rack in front of red rack and put other racks sequentially behind.
Close lid and hit ‘Auto Count’.
When samples are finished, throw entire scintillation vial contents into appropriately labeled waste container.