And back we go
- Failure of previous two ligation efforts prompted rethinking of ligation strategy. Will attempt a one-step triple ligation that will link phsABC to B0015 and insert them both into a standard single-resistance iGEM background. phsABC will be digested with EcoRI and SpeI, B0015 with XbaI and PstI, and pSB1C3 with EcoRI and PstI, allowing for them all to be ligated together.
- Needed more phsABC, so ran five PCR reactions to amplify it from plasmid pSB74. Reaction mixture contents were the same as the DMSO variant used on 6/30 as was the thermocycler protocol (phs50). The results were then run on a gel
Gel of PCR amplified phsABC Extreme right lane:1 kb ladder, all other lanes PCR reaction mixtures and their spillover
- Needed chloramphenicol plates for pSB1C3-based constructs, so mixed 350 μL of 50 μg/μL chloramphenicol with an autoclaved solution of 7.5 g agar and 12.5 g LB in 500 mL H2O and poured plates.
- Ran all three digestion reactions for 30 minutes at 37 °C before heat killing for 20 minutes at 80 °C. Digestion mixtures had the following components:
|Digestion Reaction Components
| phsABC Digestion||B0015 Digestion||pSB1C3 Digestion
|5 μL EcoRI Buffer 10x || 5 μL NEB Buffer 3 10x || 5 μL EcoRI Buffer 10x
| 0.5 μL BSA 100x
|21.1 μL phsABC soln (1 μg DNA) || 3.8 μL B0015 soln (1 μg DNA)|| 15 μL phsABC soln (375 ng DNA)
|1.8 μL EcoRI & 1.8 μL SpeI
||1.8 μL XbaI & 1.8 μL PstI
||1.2 μL EcoRI, 1.2 μL PstI, & 1.2 μL DpnI
|20.8 μL H2O || 37.1 μL H2O || 15.9 μL H2O
Wetlab work for this day is also recorded on page 51 of the hard copy lab notebook.