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

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*'''Insert:'''
*'''Insert:'''
*#Digest miniprepped KaiA/B/C in GeneArt plasmid with X-P
*#Digest miniprepped KaiA/B/C in GeneArt plasmid with X-P
-
*'''bkb:'''
+
*'''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)
*'''KaiABC ligation:'''
*'''KaiABC ligation:'''

Revision as of 13:49, 14 August 2006



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)
  • KaiABC ligation:
    1. KaiABC\X-P + J04500\S-P (stage I)
    2. KaiABC\X-P + pSB4K3 (well-formed BB)
  • 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
Click for legend
Click for legend


It appears that our vector self-ligated.
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