IGEM:Yale/2010/Notebook/2010/07/07

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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.

Prep work

  • 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
    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.

Digestions

  • 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 DigestionB0015 DigestionpSB1C3 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.

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