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| [http://karma.med.harvard.edu/mcb100/Main_Page Work in MCB100] | | [http://karma.med.harvard.edu/mcb100/Main_Page Work in MCB100] |
| [[IGEM:Harvard/2006/cyanobacteria/peng_labbook | Lab notebook from iGEM06]] | | [[IGEM:Harvard/2006/cyanobacteria/peng_labbook | Lab notebook from iGEM06]] |
| ==Notebook==
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|
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| Below are notes taken during meetings or during lab; they will be either in pdf or below depending on platform used. Let me know if you need a native OneNote copy instead.
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|
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| ===June 12th===
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|
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| '''Morning'''
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| *[[Media:ZS_brainstorm.pdf | Notes from first meeting]] (pdf)
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|
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| '''Afternoon'''
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| *Explained BioBricks Format
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| *Created 1% Agarose Gel
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| *Folding DNA nanostructures
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| *Transformation of R0010, E7104, E0241 into Top-10 Competent E. Coli
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|
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| R0010 - Lac operon promoter
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| E7104 - T7 promoter + GFP
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| E0241 - GFP
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|
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| ===June 13th===
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|
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| '''Morning'''
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| *Brainstorming by presenting previous projects
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| *[[Past_project_notes]]
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|
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| '''Afternoon'''
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| *TF's created a 1% Agarose gel which we ran our DNA scaffold samples on
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| **130V, 45m+, imaged afterwards
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| DNA Scaffold types
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| -both
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| -scaffold only
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| -oligos only
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| *Checked plating of transformant cells; no growth on control
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| *Grew transformant cells in liquid culture (5mL) overnight with 50uL ampicillin
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| *More brainstorming - Perry presented his domain swapping experiment, discussed DNA nanostructure box
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|
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| ===June 14th===
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|
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| '''Morning'''
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| *DNA Miniprep of transformant colonies
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| **Out of 5mL of liquid culture, reserved 1mL for gylcerol+freeze and 4mL other
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| **pelleted E. coli for each sample
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| **followed QIAprep Miniprep Kit for Microcentrifuge directions
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| ***Used warm dH20 for elution
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| ***Put at 40C for ~2 min to evaporate ethanol before elution
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| ***Forgot to label after elution --> don't know what is what
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| ****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.
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| ***Nanodrop demonstration
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| Nanodrop results
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| -1: 31.2ng/uL 260/280:1.75 260/230:1.92
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| -2: 38.5ng/uL 260/280:1.83 260/230:1.87
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| -3: 33.8ng/uL 260/280:1.70 260/230:1.44
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|
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| '''PROBLEM:''' Messed up labeling of the plasmids! To diagnose, ran a 15min e-Gel to find out which is R0010.
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|
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| *Digestion of vector/insert
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| **Digested R0010 (200bp cutout) as vector at S and P site.
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| ***.5uL Spe1, .5uL Pst1
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| *** 11uL h20
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| *** 2.5uL 10X BSA
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| *** 2.5uL #2 NebBuffer
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| *** 8uL DNA
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| **Digested other 2 (~900bp cutout) as insert at X and P site.
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| ***.5uL Xba1, .5uL Pst1
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| *** 11uL h20
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| *** 2.5uL 10X BSA
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| *** 2.5uL #3 NebBuffer
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| *** 8uL DNA
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| **Incubate @ 37C for 1h
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| *Phosphatase
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| **80C@15min to kill enzyme activity
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| **Used CIP (1 unit) into the R0010, 1h@37C
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| *Run on 1% agarose gel
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| *Image, Cutout, and Purify
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| **Can isolate the three from the gel
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|
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| '''Result'''
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|
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| [[Image:ZS_DR_gelimage_0614.jpg |thumb| "results of PCR"]]
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| Ladder=1kb+
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| Lane 1=R0010 (#1)
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| Lane 2=E0241 (#2)
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| Lane 3=E7104 (#3)
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|
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| ===June 15th===
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| '''Morning'''
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| *Brainstorming with Pam
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| **Infeasibility of DNA nanostructures
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| **(Linked)
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| *Conduct ligation reaction
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| **6uL insert, 2uL vector, 2uL buffer, 10uL other buffer
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| **5 min incubation @ RT
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| *Conduct transformation
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| **20mL cells for positive, negative, and exp. ea (top 10)
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| '''Afternoon'''
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| *Plate transformant cells on LB-CARB
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| *Brainstorming session in the afternoon
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| **Linked
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|
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| ===June 16th===
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| '''Morning'''
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| *Talk with Prof. Shih about DNA nanostructures and vailidity
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| **Linked
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| '''Afternoon'''
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| *Talks on two papers
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| **[[iGEM:Harvard/2006/Brainstorming_Papers_-_Zhipeng]]
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| *Other talks can be found at [[IGEM:Harvard/2006/Brainstorming]]
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|
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| ===June 17th (Sat)===
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| '''Afternoon'''
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| *Met up in the evening to conduct research on cyanobacteria with Hetmann, Dave, and Jeff
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|
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| ===June 18th (Sun)===
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| '''Afternoon'''
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| *Finalized information and created Powerpoint
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| **[[IGEM:Harvard/2006/Cyanobacteria]] for link to cyanobacteria information
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| **[[Media: Presentation.ppt]] for Powerpoint delievered (requires Beta 2007 to work correctly)
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|
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| ===June 19th (Week 2)===
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| '''Morning'''
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| *Presentation of four projects
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| **DNA nanostructures (Matt Katie Valerie Tiffany)
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| **Fusion Proteins (Perry)
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| **Universal Cell Signaling (Lewis)
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| **Cyanobacteria (Peng Hetmann David Jeff)
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| *Feedback on cyanobacteria
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| **Found two sources for our plasmids: WHOI and MIT links
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| **E. coli link may not work, but should figure out synthesis
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| ***Codon Devices can do it for $0.30/bp
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| **Prof. Alexander van Oudenaarden works with cyanobacteria, [http://web.mit.edu/biophysics/people.html]
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| '''Afternoon'''
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| *Prof. Shih's discussion on the honeycomb lattice
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| *Check plates for GFP expression
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| Two colonies on experimental (R0010+E0241)!
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| Many on +
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| None on control
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| *Grow R0010+E0241 in 5mL LB + 50uL Amp (5mg/uL orig) overnight @37
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| *Further brainstorming
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| *Emailed Eric Webb about Fedex #; Peter Weigele can give us PCC7942 on Thurs.
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|
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| ===June 20th===
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| '''Morning'''
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| *Ordered culture BC11 by Sigma
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| *Alain ordered PCC7942 and WH8104 strain from WHOI
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| **Need to look up if other strains grow faster / better to culture
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| *Need to get ahold of Prof. Knoll and vanO lab to ask about cyanobacteria culture
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| *Make glycerol stocks of R0010+E0241
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| **100uL 50% glycerol, 100uL liquid media
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| *DNA Miniprep
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| **Follow handout; had 2 5mL samples, only used one of them
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| **Tubes 0.5mL PCR; labeled in blue on top
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| *Digest for diagnosis
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| **Xba1 and Pst1
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| **#.5uL Xba1, .5uL Pst1
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| **#11uL h20
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| **#2.5uL 10X BSA
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| **#2.5uL #3 NebBuffer
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| **#8uL DNA
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| **#Incubate @ 37C for 1h
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| '''Afternoon'''
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| *Research into cyanobacteria: see [[IGEM:Harvard/2006/Cyanobacteria]] for contribution
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| *Digest gel result
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| [[Image:Digest_6-20-06.jpg |thumb| "Result from digest. Lane 7 is our reaction, and the ladder is 1kB+. Despite weak signal it looks as if the digest went okay; differences in size probably due to different restriction sites used."]]
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|
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| ===June 21st===
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| '''Morning'''
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| *Populating [[IGEM:Harvard/2006/Cyanobacteria]]
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| *Organizing shopping list/calling stores
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| '''Afternoon'''
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| *Roadtrip to Home Depot to obtain items
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| *Wrote [[IGEM:Harvard/2006/Cyanobacteria/Building_cyanobacteria_incub | Guide to building a cyanobacteria incubator]]
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| *Built incubator
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| *Will pick up PCC7942 tomorrow.
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|
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| ===June 22nd===
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| '''Morning'''
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| *Working incubator!
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| *WH8102 came in the mail with SH media
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| '''Afternooon'''
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| *Went to go pick up PCC7942 and 6803 from Peter Weigele @ MIT Building 68
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| *Made liquid culture without thiosulfate
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| *Made 6 samples
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| **PCC7942 streak w/toothpick
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| **PCC7942 single w/toothpick
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| **PCC7942 single w/o toothpick
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| **PCC6803 streak w/toothpick
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| **PCC6803 single w/toothpick
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| **Control w/ toothpick
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| *Sprayed with EtOH beforehand working area
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| *16h day / 8h night, shaking, open lid, 30C
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|
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| ===June 23rd===
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| '''Morning'''
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| *Measured light intensity with lux meter
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| **Turns out only a little region has ~4200 lux; rearranged samples
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| *Tested plastic cover; does not diffuse light
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| '''Afternoon'''
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| *General research on ideal conditions for growth
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|
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| ===June 26th (Week 3)===
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| '''Morning'''
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| *Morning meetings
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| *Made more BG11 liquid media w/ thiosulfate
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| *Made 500x Thiosulfate+Na stock sol'n
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| *Brought down solid plate recipe
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| '''Afternoon'''
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| *Checked experiments
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| **See Jeff's pictures [[IGEM:Harvard/2006/Cyanobacteria#Incubator | here]]
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| **See results for the day [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-26 | here]]
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| *Created new liquid colonies
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| **125mL BG11 liquid in ''autoclaved'' glass 500mL earlemeyer
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| **'''PCC7942 dried out!'''
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| *Question to be addressed:
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| **How do the plasmids work? DNA delivery?
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| '''Evening'''
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| *Emailed Prof. Susan Golden
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|
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| ===June 27th===
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| '''Morning'''
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| *PCR tutorial
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| **Tm = 65, y = Tm -5
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| **deltaG > -3 kCal/mol
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| '''Afternoon'''
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| *Beginning primer design
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| *Ordered PCC7942 from atcc.org
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| ===June 28th===
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| '''Morning'''
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| *Sick :(
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| '''Afternoon'''
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| *Reviewed information from Prof. Golden
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| *Growth in PCC6803!
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| *Completing primer design for KaiABC extraction
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|
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| ===July 6th===
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| '''Morning'''
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| *Rehydrated oligos that arrived (10 of them)
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| **First in 250uL dh20
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| **Then diluted to 20uM ea
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| *PCR (colony) from dried PCC7942 plate
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| **Did not work
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| *Decided to model oscillation
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|
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| ===July 7th===
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| *Recieved PCC7942
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| *PCRed again, this time with liquid culture as template
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| *2 liquid cultures made, 500mL flask w/100 mL
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| **1 w/ thiosulfate
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| **1 w/o thiosulfate
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| *2 solid cultures made
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| *Modeling
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|
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| ===Post July 7th===
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|
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| Look into our cyanobacteria page at [[IGEM:Harvard/2006/Cyanobacteria/Notebook]].
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