Team:UC Davis/Notebook/Week 2

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We spent a good chunk of this week waiting for primers so that we could begin construction. During this time, we ligated our newly made LacI promoter mutants into the promoter screening plasmid, J61002, so that we could observe possible mutations. With the arrival of some primers at the end of the week, we started construction by PCRing B0034 into E0240.
--Monday 6/20/11--

Today we nanodropped the 12 digested mutated LacI promoters and the [http://partsregistry.org/Part:BBa_J61002 J61002] promoter screening plasmid such that we could ligate them:

Sample ID ng/uL 260/280
LacI 1 188.93 1.19
LacI 2 113.96 1.26
LacI 3 87.29 1.19
LacI 4 88.23 1.20
LacI 5 109.35 1.22
LacI 6 90.64 1.18
LacI 7 536.16 1.33
LacI 8 415.75 1.33
LacI 9 462.50 1.33
LacI 10 544.12 1.36
LacI 11 461.32 1.33
LacI 12 498.83 1.35
J61002 11.92 1.95


We then ligated the 12 mutated LacI promoters into the J61002 promoter screening plasmid. We had planned to transform all of these ligations but unfortunately ran out of LB+Carb plates. We were still able to transform about 7 of the 12 ligations. We also rehydrated and transformed the B0015 terminator.

We miniprepped and digested the following parts:

  • I13458 (with EcoRI and SpeI)
  • I13453 (with EcoRI and XbaI)
  • R0010 (with SpeI and PstI)
  • R0040 (with SpeI and PstI)
  • R0051 (with SpeI and PstI)

--Tuesday 6/21/11--

After yesterday's plate shortage, we took on the new task of making new LB+Carb plates.

We also nanodropped many of the minipreps (MP) and gel purifications (GP) from earlier:

Sample ID ng/uL 260/280
J23101 MP 338.75 1.87
I13456 MP 245.92 1.86
I13453 MP 181.48 1.85
R0010 MP 115.98 1.86
R0051 MP 101.21 1.89
E0040 MP 240.71 1.82
R0040 MP 80.18 1.92
C0012 MP 165.16 1.87
R0010 GP 22.42 1.73
I13458 GP 16.63 1.86
R0040 GP 20.39 1.62
I13453 GP 15.10 1.74
R0051 GP 7.27 2.69

--Wednesday 6/22/11--

Miniprepped B0015 and made glycerol stocks of I13453, I13458, and B0015. We also made some 10 mM IPTG from more concentrated stocks.

--Thursday 6/23/11-- After waiting most of the week some of our primers finally came in! We ran PCR with our B0034+E0240 forward primer and our E0240 forward primer.
--Friday 6/24/11--

PCR purified B0034+E0240 and E0240. Digested B0034+E0240 with XbaI and PstI.

Today we digested the following parts:

  • B0034 (with SpeI and PstI)
  • C0040 (with XbaI and PstI)
  • C0051 (with XbaI and PstI)
  • C0012 (with XbaI and PstI)
  • J61002 (with EcoRI and PstI)
  • psB1K3 (with EcoRI and PstI)
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