Jessica Karen Wong/Notebook/2007-6-25
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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