Jessica Karen Wong/Notebook/2007-6-25

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