IGEM:MIT/2007/Notebook/2007-7-23

Agenda

1. Polystyrene Assay
   • Morning: get correct OD
   • Add AHL
   • Afternoon: Test
2. Digest I13500
3. Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids)
   • run overnight
4. dilute sequencing primers
   • sequence PHIE6P
   • sequence F2620+B0034+CPX
5. Order T7 antibody

Digestion of I13500

• Made two mixtures

4.5 µl I13500 (Miniprep #1, 223.5 ng/µl)
1 µl   XbaI
1 µl   PstI
0.5 µl BSA
5 µl   NEB3 Buffer
38 µl  H20
-------------------------
50 µl  Total

• Placed in 37C @ 10:20am --> Take out @ 1:20pm

Diluted BioBrick Sequencing Primers

You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room).

Diluted from 100 µM stock to 32.2 µM with total volume of 1.55 mL each.

Made 1 VF2 and 1 VR for iGEM use.

Polystyrene Assay Protocol

• Cell Lines:
  • Controls: DH5-alpha (not transformed)
  • FHUA PHIE6 --> IPTG
  • CPX --> AHL
• Media:
  • H20
  • PBS
  • PBS + Tween + BSA

• Protocol

1. Add AHL/IPTG to Liquid Culture
2. Wait an hour for AHL induced expression
3. Incubate wells in wash media for 30 minutes on rocker at speed 5
4. Centrifuge cells out of Liquid Culture media
5. Resuspend in wash media
6. Add cells to appropriate well
7. Place on rocker for 1 hour at speed 5
8. Aspirate/Pipet out media in wells