User:DavidRamos/Laboratory Notebook

Welcome to my iGem 2006 summer lab notebook!

Morning
 Plasmid Miniprep  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
 * 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

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)

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

Morning
Afternoon
 * Talk with Prof. Shih about DNA nanostructures and vailidity
 * Talks on two papers
 * iGEM:Harvard/2006/Brainstorming_Papers_-_David_Ramos
 * Other talks can be found at IGEM:Harvard/2006/Brainstorming

Evening

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

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)

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

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
 * .5uL Xba1, .5uL Pst1
 * 11uL h20
 * 2.5uL 10X BSA
 * 2.5uL #3 NebBuffer
 * 8uL DNA
 * 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!

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