July 3, 2009

Started log phase growth for BB2, 3, 4, 5 in 100 ml of LB with Amp (100 ul)
- BB2, 3, 4 appeared to be contaminated as there was no significant increase in absorbency. We suspect the source of contamination was from the pipette tip when adding the ampicillin to the LB. The tip had been used repeatedly.
- Due to the contamination observed, the ampicillin may also have been contaminated. If the same situation occurs again, it may be due to this.
Miniprepped BB5 ([] = 3.54.4 ng/ul) and froze 2 cryotubes for long term stock

July 5, 2009

2, 3, 4 overnight cultures did not grow well - replated 2, 3, 4 on new Amp plates
Gel image (~1hr @ 120 V)
- L1: Ladder - too concentrated (not well resolved) probably because gel was made with 0.5X TBE while loading buffer for gel was 1X TBE - next time use 0.5X
- L2: BB1 - not resolved, appears to not be fully digested, too much DNA? Appears to be proper plasmid, but a part ran off. Next time run gel for less time (~30 min)
- L3: BB5 - nothing appeared
- L4: C - looks very faint, add more next time (30 ul instead of 20 ul)
- K - same as C
Redigested BB5 - followed digestion protocol suggested in BioBrick Assembly Manual
- Digestion mixture: 500 ng plasmid ([BB5] = 328.0 ng/ul, used 1.5 ul), 1 ul EcoRI-HF, Spel each, 10X NE Buffer 2 5 ul, 100X BSA 0.5 ul, made up to 50 ul
After nearly 4 hours in the incubator, the tet plasmid culture had an absorbency of 0.011. We decided to leave it overnight and to miniprep tomorrow.