User:DavidRamos/Laboratory Notebook

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Welcome to my iGem 2006 summer lab notebook!

Contents

June 14th

Morning

Plasmid Miniprep

  • lac operon promoter
    • R0010
  • promoter and GFP
    • E0241
  • GFP
    • E7104
  • DNA Miniprep of transformant colonies
    • Out of 5mL of liquid culture, reserved 1mL for gylcerol+freeze and 4mL other
    • followed QIAprep Miniprep Kit for Microcentrifuge directions
      • Eluted with warm dH20
      • Put at 40C for ~2 min to evaporate ethanol before elution
      • Forgot to label after elution --> don't know what is what
        • Sol'n: During digest will have to run PCR, can tell R0010 from rest, but E7104/E0241 is only different by 30bp; if doesn't work can flip and try two experiments.
      • Nanodrop demonstration
Nanodrop results
  -1: 31.2ng/uL 260/280:1.75 260/230:1.92
  -2: 38.5ng/uL 260/280:1.83 260/230:1.87
  -3: 33.8ng/uL 260/280:1.70 260/230:1.44

PROBLEM: Messed up labeling of the plasmids! To diagnose, ran a 15min e-Gel to find out which is R0010.

  • Digestion of vector/insert
    • Digested R0010 (200bp cutout) as vector at S and P site.
      • .5uL Spe1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #2 NebBuffer
      • 8uL DNA
    • Digested other 2 (~900bp cutout) as insert at X and P site.
      • .5uL Xba1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #3 NebBuffer
      • 8uL DNA
    • Incubate @ 37C for 1h
  • Phosphatase
    • 80C@15min to kill enzyme activity
    • Used CIP (1 unit) into the R0010, 1h@37C
  • Run on 1% agarose gel
  • Image, Cutout, and Purify
    • Can isolate the three from the gel

Result

"results of PCR"

 Ladder=1kb+
 Lane 1=R0010 (#1)
 Lane 2=E0241 (#2)
 Lane 3=E7104 (#3)

June 15th

Morning

  • Brainstorming with Pam
  • Conduct ligation reaction
    • 6uL insert, 2uL vector, 2uL buffer, 10uL other buffer
    • 5 min incubation @ RT
  • Conduct transformation
    • 20mL cells for positive, negative, and exp. ea (top 10)

Afternoon

  • Plate transformant cells on LB-CARB
  • Brainstorming session in the afternoon


June 16th

Morning

  • Talk with Prof. Shih about DNA nanostructures and vailidity

Afternoon

June 17th (Sat)

Evening

  • Worked remotely from New York on cyanobacteria presentation with Peng, Hetmann, and Jeff

June 18th (Sun)

Afternoon

June 19th (Week 2)

Morning

  • Presentation of four projects
    • DNA nanostructures (Matt Katie Valerie Tiffany)
    • Fusion Proteins (Perry)
    • Universal Cell Signaling (Lewis)
    • Cyanobacteria (Peng Hetmann David Jeff)
  • Feedback on cyanobacteria

Afternoon

  • Prof. Shih's discussion on the honeycomb lattice
  • Need to design easy data structures for Python program
  • Check plates for GFP expression
 Two colonies on experimental (R0010+E0241)!
 Many on +
 None on control
  • Grow R0010+E0241 in 5mL LB + 50uL Amp (5mg/uL orig) overnight @37
  • Further brainstorming

June 20th

Morning

  • Dive-Into-Python Chapters 2-5
    • Functions, Datatypes, List functions, Classes and Objects
  • Make glycerol stocks of R0010+E0241
    • 100uL 50% glycerol, 100uL liquid media
  • DNA Miniprep
    • Follow handout; had 2 5mL samples, only used one of them
    • Tubes 0.5mL PCR; labeled in blue on top
  • Digest for diagnosis
    • Xba1 and Pst1
      1. .5uL Xba1, .5uL Pst1
      2. 11uL h20
      3. 2.5uL 10X BSA
      4. 2.5uL #3 NebBuffer
      5. 8uL DNA
      6. Incubate @ 37C for 1h

Afternoon

  • Ran DNA on an E-gel. Lane7 in the picture below:

Image:Digest 6-20-06.jpg

  • Further brainstorming on DNA nanostructure design.
    • Considered flat-sheet and honeycomb-lattice versions of each of the following structures: cylinder, tetrahedron, cube.
    • Eventually decided to go with honeycomb-lattice cylinder since we have already made those.
    • Considered multiple ideas for DNA nanotube lids. Measurements and calculations will be posted tomorrow.
    • Peng did some research and found that we need to set up some equipment to raise cyanobacteria. And by set up, I mean build. Home Depot road trip on the morrow? Stay tuned!

June 21st

Morning

  • Figured out what supplies we needed for cyanobacteria growing with Peng.
    • Shopping list can be found here.

Afternoon

  • Went shopping! Home Depot ftw.
  • Installed the fluorescent light on the incubator.
Incubator exterior
Incubator exterior
Incubator interior
Incubator interior
Incubator interior (closeup)
Incubator interior (closeup)


  • How many Harvard iGem students does it take to screw in a light bulb?
    • Answer: 3, a TF, and 2 hours.
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