User:DavidRamos/Laboratory Notebook

Welcome to my iGem 2006 summer lab notebook!

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

 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

• Talks on two papers
  • iGEM:Harvard/2006/Brainstorming_Papers_-_David_Ramos
• Other talks can be found at IGEM:Harvard/2006/Brainstorming

June 17th (Sat)

Evening

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

June 18th (Sun)

Afternoon

• Finalized information and created Powerpoint
• Half-Life
  • IGEM:Harvard/2006/Cyanobacteria for link to cyanobacteria information
  • Media: Presentation.ppt for Powerpoint presentation (requires Beta 2007 to work correctly)

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:

• 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 interior

Incubator interior (closeup)

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