Jessica Karen Wong/Notebook/2007-6-25

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Contents

To Do

  • If primers are in PCR I2055 and redo's of T9002 and E0240
  • Transform ligations of R-J-AT3, R-J-AC3, B-J-AT3, B-J-AC3
  • Miniprep and Digest Backbone
  • Make Glycerol stocks of Backbone

Glycerol

Made glycerols of P1010 1AK3, P1010 1AC3, P1010 1AT3, and P1010 3K3

  • Added 1ml overnight culture to 1ml 40% glycerol
  • Not sure of the strain, most likely mg1655
  • May need to remake 3K3 b/c concentration was too low

Miniprep

  • First pelleted the DNA - 1.5ml of culture per eppendorf, 3 total per strain
  • Spin at 5k for 5 min then remove supernatant and then combine
  • Then follow protocol in Qiaprep miniprep handbook using 30ul to elute the DNA

Transformation

  • See protocol
  • Transformed RFP RBS Tester (blue) and RFP Promoter Tester (green) with mg1655
  • Plated each on both Tet and Chlor plates

Digest

  • Digested backbones with E and P
  • Concentrations of the plasmids:
    • 1AC3 214.8 ng/ul used 5ul
    • 1AK3 235.7 used 5ul
    • 1AT3 232.4 used 5ul
  • Ran overnight

3K3 had a very low concentration, we minipreped the rest of the culture that we used to make the glycerols but was still low Made a new overnight of 3K3

PCR

  • Did a 100ul preparatory PCR of I2055
  • Primer dilution: I0255F 34.19nmol added 855ul of water to get 40uM, I2055R 18.19 nmol added 455ul
  • Ran overnight
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