Swartz:Protocols/Cell Free

https://docs.google.com/document/d/1PVxGVnLibUXHoWzrOPR8Sx9j6kVNHJnpLg15NIx5Wx0/edit

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.