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 18:31, 14 August 2006
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To-do
- Midiprep pSB4A, J04500 (online instructions here, page 16)
- Insert:
- Digest miniprepped KaiA/B/C in GeneArt plasmid with X-P
- Backbone:
- Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy)
- Find and transform B0034
- KaiABC ligation:
- KaiABC\X-P + J04500\S-P (stage I)
- KaiABC\X-P + pSB1AK3 (well-formed BB)
- KaiABC\X-P + B0034\S-P (stage I)
- Sequencing:
- Sequence miniprepped KaiA/B/C + GA vectors, natural KaiA
- Western blots:
- Ligate GFP dev and pSB4A3
- Inoculate GFP dev + pSB1A2
E-gel images of colony PCR of J04500 + KaiA/B/C
It appears that our vector self-ligated.
