4 September 2006 – John Welsh
- 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.
Spotting and washing samples:
- 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.
Preparing samples for scintillation counter:
- 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.