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

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==To-do==
==To-do==
*'''Midiprep'''
*'''Midiprep'''
*# Midiprep pSB4A3, J04500 (online instructions [http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_Plasmid/1034637_HB_QIAGENPlasmid_112005.pdf here], page 16)
*# Midiprep pSB4A3, J04500 (online instructions [http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_Plasmid/1034637_HB_QIAGENPlasmid_112005.pdf here], page 16) ''(in progress)''
*'''Insert:'''
*'''Insert:'''
*#Digest miniprepped KaiA/B/C in GeneArt vector with X-P ''(in progress; 2 hr digest finishes at 7PM, 4 hr finishes at 9PM)''
*#Digest miniprepped KaiA/B/C in GeneArt vector with X-P ''(in progress; 2 hr digest finishes at 7PM, 4 hr finishes at 9PM)''
Line 31: Line 31:
*'''Western blots:'''
*'''Western blots:'''
*#Ligate GFP dev\X-P and pSB4A3\S-P, transform ''(Wait until we've midiprepped more pSB4A3 and do another digest, because the pSB4A3\S-P from our digest assay was a curious size)''
*#Ligate GFP dev\X-P and pSB4A3\S-P, transform ''(Wait until we've midiprepped more pSB4A3 and do another digest, because the pSB4A3\S-P from our digest assay was a curious size)''
*#Inoculate GFP dev + pSB1A2  
*#Inoculate GFP dev + pSB1A2


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

Revision as of 21:25, 14 August 2006

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To-do

  • Midiprep
    1. Midiprep pSB4A3, J04500 (online instructions here, page 16) (in progress)
  • Insert:
    1. Digest miniprepped KaiA/B/C in GeneArt vector with X-P (in progress; 2 hr digest finishes at 7PM, 4 hr finishes at 9PM)
    2. Gel-purify
    3. (maybe) Inoculate more KaiA/B/C in GA vector
  • Backbone:
    1. Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy) (in progress; 2 hr digest finishes at 7PM, 4 hr finishes at 9PM)
    2. CIP treatment of the above
    3. Gel-purify
    4. Find and transform B0034
  • KaiABC ligation:
    1. KaiABC\X-P + J04500\S-P (stage I)
    2. KaiABC\X-P + pSB1AK3 (well-formed BB) (we can purify this backbone from the J04500\X-P digest)
    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\X-P and pSB4A3\S-P, transform (Wait until we've midiprepped more pSB4A3 and do another digest, because the pSB4A3\S-P from our digest assay was a curious size)
    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

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


Add 5.5 µL of the corresponding master to each tube.

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