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.

Absorbency of tet plasmid this morning = 0.62
- Made 2 cryotubes of stock (put in -80 freezer), miniprepped [] = 56.4 ng/ul
After 30 minutes of running the gel at 97 mV, the bands could be seen but there was not enough separation. We continued to run it.
Started overnight culture for BB2, 3, 4 in 100 ul, 130 ul, and 100 ul of LB
After 1h 45min, gel was examined:
- Comparing lanes 2 and 3 (BB1 digested vs. undigested), we conclude that BB1 is the correct size and results of digested are as expected
- Lanes 4, 5 had very faint bands. Since they were digests of BB5 done on Sunday and repeated Monday, we conclude the digestion time may be too long such that most DNA had been degraded
- Lane 6 was undigested BB5; the band shows the DNA sample was not degraded
- Lane 7 and 8 were reruns of C and K plasmids. Although bands were faint, they were the size expected
- Future adjustments:
- 2% agarose was too thick because after running for nearly 2 hr, the separation did not happen. Will try 1.5% agarose next time
- Digestion time for BB5 should be shortened

Started log phase growth for BB2, 3, 4
Transfected DB3.1 cells with BB6 (R0040) and the operation vector (pSB3C5) and plated them on appropriate antibiotic plates
Miniprepped BB2, 3, 4 from overnight culture
BB2, 3, 4 stocked in cryotubes with glycerol from log phase culture
Poured 8 Amp plates

Digested BB2, 3, 4, tet, and 5 for 1h and 3h
Plated BB1, 5, C, K from stock tubes
Transfected cell plates from yesterday did not show any colonies. We therefore plated the remaining liquid culture (stored at 4 degrees Celsius) onto appropriate antibiotic plkates
Ran a gel of the digests
- Loaded 20 ul of sample in each well
- Used all of 50 ml agarose + TBE to make gel to make deep wells
- 1h digests were kept on ice while waiting for 3h digests
- DNA ladder was heat shocked at 60 degrees Celsius for 3 min before mixing with dye and TBE

Gel turned out well though there are still problems with the ladder - suggest loading small amount of DNA ladder in a small well
There is a minimal difference between 1h and 3h digests - suggest we stick with 1h digest in the same conditions. Also, future - if there is a free well, try 30 min digest

All 4 replates from yesterday grew colonies (1, 5, C, K), indicating our stock tubes do indeed contain the plasmids
Replated BB2, 3, 4, Tet from stock tubes
Digested BB1, 2, 5 and ran on 1.3% TAE gel with 100 bp DNA ladder
- 10 min and 1h digests don't show a big difference and we should try a 10 min digest in future
- BB1 appears to have contaminations as the top band was not at expected location
- BB5 did not appear on the gel again. We suspect the plasmids are contaminated with DNase or other proteins. We will run another gel with undigested BB5 after incubating @ 37 C on Monday
- The small fragments of the biobricks were not seen on the gel. Austin said this was expected.

=July 13, 2009

Replates of BB2, 3, 4, Tet are growing well
Started overnight culture of BB6 and 0 with antibiotics
Transfected DH5-a cells with BB7, BB1, 5 and TM0785 and plated on Amp plates (2 plates for each BB)