IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-14

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To-do

  • Midiprep pSB4A, J04500 (online instructions here, page 16)
  • Insert:
    1. Digest miniprepped KaiA/B/C in GeneArt vector with X-P
  • Backbone:
    1. Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy)
    2. Find and transform B0034
  • KaiABC ligation:
    1. KaiABC\X-P + J04500\S-P (stage I)
    2. KaiABC\X-P + pSB1AK3 (well-formed BB)
    3. KaiABC\X-P + B0034\S-P (stage I)
  • Sequencing:
    1. Sequence miniprepped KaiA/B/C + GA vectors, natural KaiA
  • Western blots:
    1. Ligate GFP dev and pSB4A3
    2. Inoculate GFP dev + pSB1A2

E-gel images of colony PCR of J04500 + KaiA/B/C

Click for legend
Click for legend


It appears that our vector self-ligated.

Digestion Assay for 8/14

Add 5.5 µL of the corresponding master to each tube.
Label DNA Amount Water Enzymes Amount NEB buffer BSA (100x) Time Master Total
Digests 1 J04500 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 2 hrs 1 25 µL
2 J04500 19.5 µL 1.75 µL SpeI & PstI 0.5 µL, 0.5 µL Buffer 2 (2.5 µL) 0.25 µL 2 hrs 2 25 µL
3 J04500 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 4 hrs 1 25 µL
4 J04500 19.5 µL 1.75 µL SpeI & PstI 0.5 µL, 0.5 µL Buffer 2 (2.5 µL) 0.25 µL 4 hrs 2 25 µL
5 KaiA 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 2 hrs 1 25 µL
6 KaiB 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 2 hrs 1 25 µL
7 KaiC 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 2 hrs 1 25 µL
8 KaiA 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 4 hrs 1 25 µL
9 KaiB 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 4 hrs 1 25 µL
10 KaiC 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 4 hrs 1 25 µL
Master Mixes 1 - - 15.75 µL XbaI & PstI 4.5 µL, 4.5 µL Buffer 3 (22.5 µL) 2.25 µL - - 49.5 µL
2 - - 7 µL SpeI & PstI 2 µL, 2 µL Buffer 2 (10 µL) 1 µL - - 22 µL
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