"
Team:Cambridge/Templates/HybridBook
From 2010.igem.org
| Line 117: | Line 117: | ||
{{:Team:Cambridge/prototypeLab}} | {{:Team:Cambridge/prototypeLab}} | ||
<html></td></tr></table> | <html></td></tr></table> | ||
| - | + | </div></div></html> | |
{{:Team:Cambridge/Templates/footerMinimal}} | {{:Team:Cambridge/Templates/footerMinimal}} | ||
Revision as of 22:30, 18 August 2010
Viewing project diary
View protocols and data
|
Week 1: Monday 12th - Sunday 18th July
Monday{{:Team:Cambridge/Templates/{{{src}}}|src=Cambridge-Postit.jpg}} We spent the morning compiling a large set of ideas, each on its own post-it-note. We categorised them and excluded those that were clearly unfeasible. If you like, you can see the long list. In the morning we narrowed them down to a shortlist of projects.
Tuesday We spent the morning researching the shortlisted projects and seeing a demonstration of the Techne PCR machine by Bibby Scientific. In the afternoon we worked on a practical with Qiagen equipment, helped by Simon Morgan. We miniprepped DNA, and then ran it on a gel. Wednesday In the morning we looked at past iGEM projects and compiled characteristics of successful projects, this was a very useful exercise. We also presented the topics we had researched in detail to each other which gave us a better sense of which areas we wanted to pursue further.
We created pages for each project idea:
Thursday We discussed quiescence in the morning, in particular the possibility of IP issues. We concluded we should consult David Summers who holds patents in the area. We decided there was not enough for a full project in the topic. We also considered whether altering RNA structure was a realistic aim for iGEM. Bio-Luminescence and Quiescence joint project - A functional breakdown of the project was proposed:
We discussed finding a "super" luciferase with 12x the affinity for the substrate and not for the product. The firefly systems are all eukaryotic, so we suggested that we could perhaps do this in yeast. Control of quiescence could also occur via cyclin control in yeast.
Friday
Bill and Will attempted to estimate the luminosity of a bacterial colony, and compare this to the lower threshold of the human eye during scotopic vision. They used an estimate for the eye sensitivity using [1]. Theo predicted the structure of Rcd using mFold, but got a different conformation to the one shown in the paper, perhaps the algorithms used on the program have changed between now and when the paper was written. We listened to Fireflies by Owl City and concluded the lyrics make no sense. SaturdayPeter made a physical model of the X RNA to visualise the structure Sunday
|
Tuesday1. Experiment: Streaking out of bacterial cultures (Peter & Anja)On LB agar plates:
On LB agar + kan plates:
Incubated at 37°C overnight Wednesdayall strains grew with individual colonies. 2. Experiment: Set up overnight culture of TOP10 (Emily & Anja)Inoculated single bacterial colony (ATP10) in 12ml LB, incubated at 37°C with shaking (180 rpm) overnight. ThursdayInoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am OD600 measurements:
3. Preparation: CCMB80 Buffer (Volume500ml)Arrows label dilutions to the indicated concentration
4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight Friday5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare) Inoculated 800ml SOB with 3ml of TOP10 overnight culture (grown from single colony), incubated at 37°C with shaking (180rpm) Put on at 9.45am OD600 measurements:
Cooled cells and CCMB80 buffer in ice bath for around 20min. Aliquoted 600ml culture in 12x50ml centrifuge tubes (pointed bottom). Centrifuged at 3000g at 4°C for 10min. Discarded supernatatent. Gently resuspended cell pellet in 20ml ice cold CCMB80 buffer per tube. Incubated on ice for 20min. Centrifuget at 3000g at 4°C for 10min. Pooled Pairs of tubes together. 12 tubes ---> 6tubes. Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C. Saturday & Sunday6. Experiment: Measuring competency of TOP10 (Fernan)TOP10 coopmetent cells (cc) taken out of -80°C freezer and put on ice. Check for thawing ater 5minutes (leave on ice). Cut 1ml pipette tips with scissors sterilised in ethanol and flamed with a Bunsen burner. Transfer ~50μl of TOP10 cc into 1.5ml Eppendorf tube with cut pipette tip. (will have to judge ~50μl by eye). Added 1μl of pUC19 (standard control plasmid) at 10-5μg/μl. Held on ice for 30min. Heat shockede for 60s at 42°C (waterbath). Put on ice for ~2min. Added 250μl pre-warmed (in 37°C incubator) SOC. Incubated at 37°C for 1h with rotation (for this purpose stick Eppendorf tubes in 12ml falcon tubes and put tape over the top). Plated 20μl on LB agar paltes with Amp (with blue shaped spreader). Grow colonies overnight at 37°C.
|
