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Team:Brown/Notebook/October8
From 2010.igem.org
Friday, October 8, 2010
Miniprep of LacI-RFC25 in pSB1C3
Overnight cultures of LacI-RFC25-pSB1C3 in LB+Chlor were recovered and miniprepped. 5 of the 6 overnights were miniprepped in parallel
Key: Ligation/digest 1,2 -> overnight 1.1,1.2,1.3,2.1,2.2,2.3 -> miniprep 1.1,1.3,2.1,2.2,2.3
Nanodrop results:
- 1.1 - 68.8ng/ul
- 1.3 - 88.1ng/ul
- 2.1 - 148.3ng/ul
- 2.2 - 111.3ng/ul
- 2.3 - 67.7ng/ul
Notes - Made 2 glycerol stocks of LacI-RFC25-pSB1C3 XL1B and placed in -80.
Overnight Cultures
Went back and looked at the 10 μl transformation of Tat-L/SB1C3 from 10/4; 3 colonies of moderate size turned up.
- Colonies still visible after 3 days at room temperature.
- Picked overnights, using 1.5 mL of LB and 12 μg/mL chloramphenicol.
- Also included overnight control of random bacteria to test chloramphenicol efficacy; started incubation at 1:00 PM.
- Took 20μl Tat-L/SB1C3 transformation (10/5) and placed in 37°C gain to see if colonies similarly emerge.
Checked plates in incubator. No growth on Kan+ Bistable. No growth on PLacMnt/J06702. Maybe PLac/Mnt is not in the vector at all?
- Re-eluted Bistable part from filter paper (all)
- Transformed CI/TerTer ligation into XL1 Amp+ (at 6:20 PM)
- Transformed Bistable (in DH5α) at 7:20 PM
- Overnight culture of 3 pTetR+RBS+LacI+terter colonies (7:20 PM)
Miniprep
Miniprepped Chlor+ overnights
- Jen: 8 total. Little AraC 1-4, AraC B 1-4.
- Lily: J13002 LacI in pSB1C.
All eluted in 40μl Buffer EB.
