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

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It appears that our vector self-ligated.
It appears that our vector self-ligated.
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==Digestion Assay for 8/14==
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|
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| align="center" style="background:#f0f0f0;"|'''Label'''
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| align="center" style="background:#f0f0f0;"|'''DNA'''
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| align="center" style="background:#f0f0f0;"|'''Amount'''
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| align="center" style="background:#f0f0f0;"|'''Water'''
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| align="center" style="background:#f0f0f0;"|'''Enzymes'''
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| align="center" style="background:#f0f0f0;"|'''Amount'''
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| align="center" style="background:#f0f0f0;"|'''NEB buffer'''
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| align="center" style="background:#f0f0f0;"|'''BSA (100x)'''
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| align="center" style="background:#f0f0f0;"|'''Time'''
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| align="center" style="background:#f0f0f0;"|'''Master'''
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| align="center" style="background:#f0f0f0;"|'''Total'''
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|-
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| 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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| ||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
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|-
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| 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
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|-
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| ||2|| - || - ||7 µL||SpeI & PstI||2 µL, 2 µL||Buffer 2 (10 µL)||1 µL || - || - ||22 µL
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|-}

Revision as of 15:41, 14 August 2006



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
Click for legend
Click for legend


It appears that our vector self-ligated.

Digestion Assay for 8/14

Label DNA Amount Water Enzymes Amount NEB buffer BSA (100x) Time Master Total
Digests1J0450019.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 2 hrs 1 25 µL
2J0450019.5 µL1.75 µLSpeI & PstI0.5 µL, 0.5 µLBuffer 2 (2.5 µL)0.25 µL 2 hrs 2 25 µL
3J0450019.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 4 hrs 1 25 µL
4J0450019.5 µL1.75 µLSpeI & PstI0.5 µL, 0.5 µLBuffer 2 (2.5 µL)0.25 µL 4 hrs 2 25 µL
5KaiA19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 2 hrs 1 25 µL
6KaiB19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 2 hrs 1 25 µL
7KaiC19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 2 hrs 1 25 µL
8KaiA19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 4 hrs 1 25 µL
9KaiB19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 4 hrs 1 25 µL
10KaiC19.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 4 hrs 1 25 µL
Master Mixes1 - - 15.75 µLXbaI & PstI4.5 µL, 4.5 µLBuffer 3 (22.5 µL)2.25 µL - - 49.5 µL
2 - - 7 µLSpeI & PstI2 µL, 2 µLBuffer 2 (10 µL)1 µL - - 22 µL