Notebook: Week 1
Monday
- Completed the required training videos for wetlab work:
http://www.yale.edu/ehs/onlinetraining/BiosafetyPart1/BiosafetyPart1.htm
http://www.yale.edu/ehs/onlinetraining/BiosafetyPart2/BiosafetyPart2.htm
http://info.med.yale.edu/chemsafe/www.yale.edu/ehs/onlinetraining/hazwaste/chemicalwaste.htm
- Received shipment of the 2011 distribution of biobricks. Identified location of the 2010 distribution.
- Worked on the project presentation to our sponsors.
- Made media for two sleeves of plates, one sleeve with Kan antibiotic added, the other with Amp. 500mL makes one sleeve. To a flask was added 12.5g of LB mix, 7.5g agar, and 500mL of water. Mixture was stirred then autoclaved on the liquid cycle for 50 minutes. The mixture was cooled to 42C and 0.5mL of the appropriate 1000x antibiotic was added. Media was poured into the plates and left to sit overnight. Antibiotics used were left over from last year (protocol is on openwetware.org)
Tuesday
- Requested AFP-eGFP generator BBa_K193209 to be sent from the registry (it was not in the 2010/11 shipments). This part was made by Tokyo Tech in 2009. It is the only AFP part that has been submitted to the registry. It consists of a T7 promoter regulated by lacI, a strong RBS, and eGFP fused to the TmTHP gene with a his purification tag attached. This part is incompatible with RFC10 (also 12, 21, 23, and 25) so we may need to use the quick change protocol. The serial cloner program was downloaded and used to identify all of the restriction sites in this part. The AFP sequence in the fusion brick is slightly truncated on both ends compared to the AFP sequence in BBa_K193201 (AFP + RBS), and compared to the sequence from the original papers isolating this gene. Also, in the fusion protein the start codon ATG does not directly come after the RBS - it goes RBS-tactagag (linker) - atatacatatg...
- Sent email to Fikrig laboratory at the Yale School of Medicine requesting use of a plasmid. This lab recently identified and cloned a novel antifreeze glycoprotein in Ixodes scapularis and showed that it enhanced survival in the cold. Perhaps they will let us use it.
- The plan is to do a proper characterization, for the first time, of the Tokyo Tech plasmid (flourescence, SDS/western, ice recrystallization activity/morphology, etc). But because we are not totally confident with how the Tokyo Tech plasmid was assembled, we may look into purchasing an AFP sequence (not from the same organism) and do some parallel experiments. Plan to meet with our advisor on West Campus for suggestions as to what sequences we should put on the AFP.
Wednesday
- The following eight parts in the Spring 2011 registry may be needed for our experiments: BBa_B0015, BBa_J32015, BBa_I712074, BBa_K103006, BBa_K12550, BBa_K093012, BBa_B0030, and BBa_I746200. We identified the plate and well location of all of these parts, as well as the resistance, and did a bacterial transformation in DH5-alpha cells.
- Competent DH5-alpha cells were thawed on ice, and the transformation tubes were also chilled on ice. 10 microlitres of autoclaved H2O was added to each well containing the DNA that we were planning to transform. 50 microlitres of cells were added to the pre-chilled 15mL culture tubes. 1 micro-litre of the DNA+H2O was added to the cells and mixed by pipetting. Tubes were left on ice for thirty minutes. Tubes were then heat-pulsed in a 42C water bath for 45 seconds, then incubated on ice for 2 minutes. Finally, 450 microlitres of LB was added to each tube and incubated for an hour at 37C on a shaker. 100 microliters of each culture was added to each plate and incubated overnight at 37C.
- Had meeting with Nigel and Catherine and one of the advisers. He showed us which benches and desks to use, where he would like us to run and image gels, the fridge and freezer space, etc.
- A doodle was created to find a time that worked for everyone for group meetings.
Thursday
- All cultures have bacterial colonies. They were placed in the fridge.
- Purchased miniprep kit, pipet tips, eppendorf tubes, round bottom culture tubes, and plates from the storeroom.
- 5mL of LB and 5 microlitres of the appropriate antibiotic (antibiotics are 1000x) were placed in culture tubes. Colonies were added with fire-sterilized materials. We picked 2 colonies for each strain, just in case. Colonies were incubated at 37C shaking overnight.
Friday
- Miniprep of the 16 cultures. Cells were spun at 5,000 G / 46,000 rpm for 20 minutes to pellet the cells. Quiagen Plasmid DNA Purification using the QIAprep Spin Miniprep kit and microcentrifuge was used. See protols. DNA was resuspended in 50 microlitres of ddH2O.
- Used NanoDrop to analyze plasmid concentration and purity. Concentrations for samples 1-16 ranged from 30ng/ul to 100ng/ul. Link to results: File:110520nanodrop.xls
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