Swartz:Protocols/Cell Free

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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.

Cell-free reaction:

  • 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.