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

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(Polystyrene Assay Protocol)
(Polystyrene Assay Protocol)
Line 49: Line 49:
#Add AHL/IPTG to Liquid Culture
#Add AHL/IPTG to Liquid Culture
#Wait an hour for AHL induced expression
#Wait an hour for AHL induced expression
-
#Incubate wells in desired media for 30 minutes on rocker at speed 5
+
#Incubate wells in wash media for 30 minutes on rocker at speed 5
-
#Centrifuge cells out of media
+
#Centrifuge cells out of Liquid Culture media
#Resuspend in new media
#Resuspend in new media
#Add cells to appropriate well
#Add cells to appropriate well
#Place on rocker for 1 hour at speed 5
#Place on rocker for 1 hour at speed 5
#Aspirate out media in wells
#Aspirate out media in wells

Revision as of 12:29, 23 July 2007

Contents

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
4.5µl I13500(Miniprep #1, 223.5ng/µl)
1µl   XbaI
1µl   PstI
0.5µl BSA
5µl   NEB3 Buffer
38µl  H20
-------------------------
50µl  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 100µM stock to 32.2µM with total volume of 1.55mL 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 new media
  6. Add cells to appropriate well
  7. Place on rocker for 1 hour at speed 5
  8. Aspirate out media in wells
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