Difference between revisions of "IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16"

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Revision as of 06:19, 17 August 2006

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To-do

  • Western blots
    • Inoculate several cultures of GFP dev. for Western blotting this afternoon
    • Take regular measurements of culture OD
    • Western blot (delayed until tomorrow because we don't seem to have GFP)
  • Construct creation
    • Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there
    • Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday
      • Digest a large amount of KaiA/B/C\X-P
      • Gel-purify
    • RBS + KaiC construct
      • Miniprep the B0034 cultures
      • Digest B0034\S-P
      • Gel-purify
      • Ligate KaiC\X-P + B0034\S-P
      • Transform KaiC\X-P + B0034\S-P

Miniprep of B0034

Title says it all; 2 60uL samples of B0034 in the freezer, and glycerol stock.

Inoculation R0010 + E0241

10 inoculations of the R0010 + E0241 were made:

  • 10 mL LB amp & 1 ul R0010 + E0241
  • 10 mL LB amp & 1 ul R0010 + E0241 (IPTG - not yet added)
  • 10 mL LB amp & 8 ul R0010 + E0241
  • 10 mL LB amp & 8 ul R0010 + E0241 (IPTG - not yet added)
  • 10 mL LB amp & 64 ul R0010 + E0241
  • 10 mL LB amp & 64 ul R0010 + E0241 (IPTG - not yet added)
  • 10 mL LB amp & 256 ul R0010 + E0241
  • 10 mL LB amp & 256 ul R0010 + E0241 (IPTG - not yet added)
  • 10 mL LB amp & 4.1 mL R0010 + E0241
  • 10 mL LB amp & 4.1 mL R0010 + E0241 (IPTG - not yet added)

Midiprep of KaiABC

The KaiABC genes were eluted (redissolved) in 200 mL of H2O.

  • KaiA: 279.1 ng/uL
  • KaiB: 285.2 ng/uL
  • KaiC: 409.7 ng/uL + 409.5 ng/uL

Ligation test and replating

We had 18 colonies grow up; see the image at the right. For each of these, I plated and did a colony PCR to test for the insert. For the first 8, I also innoculated, though it may not work.

Plates 81606.jpg

Each reaction:

  • 8 µL PCR Supermix
  • 1 µL VF2 primers @ 2uM
  • 1 µL VR primers @2uM
  • colony or 1 uL template or nothing

19 RXNS: (18 + 1 pos, which is B0034)

  • 8*20=160uL supermix
  • 1*20 = 20uL VF2
  • 1*20 = 20uL VR

PCR schedule:

  • 95C for 15'00
  • Do 30 times:
    • 95C for 0'30
    • 55C for 0'30
    • 72C for 2'00
  • 72C for 10'00
  • 4C forever


Results:

I'm confused.... the + control is B0034 (the RBS), for reference; the actual coding sequence is i think 33bp or something really short, so basically add on the size of the band in the gel to the expected KaiA,B,and C sizes.

EDIT: Actually, the previous ligation was not a self-ligation like thought. Look at http://www.openwetware.org/wiki/Image:2006_08_14_egel2.jpg, we expect a 550 bp band (like + control) if it self-ligated but it actually is a 300bp band...

EXPECTED

  • KaiA from VF to VR2: 855bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 1.4kb
  • KaiB: 309bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 859bp
  • KaiC: 1560bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 2110bp
  • B0034 Positive: 250bp (VF2-VR) = 250bp
  • Nothing: 536bp
click for legend
click for legend


Innoculation of GFP Device from Nick's Plate

4 Innoculations were made of GFP (R0010 + E0241) from Nick's bright green plate. The cells could be dead, though, so no guarantees. All 4 innoculations were made in 10mL LB + Amp. Also made a negative control of 10mL LB + Amp.

Digest of Kai\X-P and B0034\S-P

Plasmid Enzymes Incubation temperature Incubation time
KaiA in GA X, P 37C 16 hrs
KaiB in GA X, P 37C 16 hrs
KaiC in GA X, P 37C 16 hrs
B0034 in pSB1A2 S, P 37C 16 hrs
KaiA in GA X, P 45C 16 hrs
KaiB in GA X, P 45C 16 hrs
KaiC in GA X, P 45C 16 hrs
B0034 in pSB1A2 S, P 45C 16 hrs

Kai\X-P reactions

  • 8 µL midiprepped DNA
  • 2.5 µL NEBuffer 3
  • 0.5 µL XbaI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA
  • 13.25 µL H2O

Kai\X-P master mix

  • 17.5 µL NEBuffer 3
  • 3.5 µL XbaI
  • 3.5 µL PstI
  • 1.75 µL 100x BSA
  • 92.75 µL H2O

Pipette 17 µL into each reaction.

B0034\S-P reactions

  • 21.25 µL DNA
  • 2.5 µL NEBuffer 2
  • 0.5 µl SpeI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA

B0034\S-P master mix

  • 10 µL NEBuffer 2
  • 2 µL SpeI
  • 2 µL PstI
  • 1 µL 100x BSA
Pipette 3.75 µL into each reaction.