IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-14: Difference between revisions

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*'''Backbone:'''
*'''Backbone:'''
*#Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy)
*#Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy)
*#Find and transform B0034
*'''KaiABC ligation:'''
*'''KaiABC ligation:'''
*#KaiABC\X-P + J04500\S-P (stage I)
*#KaiABC\X-P + J04500\S-P (stage I)

Revision as of 11:31, 14 August 2006

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To-do

  • Midiprep pSB4A, J04500 (online instructions here, page 16)
  • Insert:
    1. Digest miniprepped KaiA/B/C in GeneArt plasmid 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.

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