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Team:TorontoMaRSDiscovery/Notebook/June
From 2009.igem.org
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June 3, 2009
- Plasmid transformed = pSB1AC3 (TEST)
- Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.
June 5, 2009
- Tet plates made
- Recipe for 200 ml (approx. 10 plates):
- 2.2 g agar in 200 ml fresh LB
- Note: do not re-autoclave LB, it will caramelize!
- Recipe for 200 ml LB:
- a) 1 g yeast extract
- b) 2 g peptotryptone
- c) 2 g NaCl
- d) 200 ml water
- Recipe for 200 ml (approx. 10 plates):
- Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
- Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
- Swirl and poured into prepared, labeled plates
- Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
- Inverted and put in 37 degree incubator to dry
June 8, 2009
- Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
- Bacterial liquid culture placed in shaker at 10:51 a.m.
June 9, 2009
- Digested miniprepped gel with EcoRI and SpeI
- Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
- DNA Ladder made - 6 microlitres of stock used
June 10, 2009
- Poured 10 Tet plates following procedure on June 5, 2009
- Due to problems with DNA ladder and restriction enzyme digest yesterday, procedures were followed again today with the following adjustments
- DNA was diluted and run on lanes 1-5 of gel:
- Lane 1 - 1X
- Lane 2 - 1/6X
- Lane 3 - 1/36X
- Lane 4 - 1/10X
- Lane 5 - 1/100X
- Restriction enzyme digest was repeated with an incubation time of 4 h at 37 degrees Celsius and heat shock at 80 degrees Celsius was done in hot water bath
- Results: Dilution did not work well (ladder could not be visualized). Digest appeared to work as 3 bands could be seen in last 2 lanes
- Adjustments for tomorrow:
- Spin down enzymes before using
- Overnight digest
- DNA was diluted and run on lanes 1-5 of gel:
June 11, 2009
- We did not have enough isolated plasmid DNA to justify doing another gel so we decided to perform another gel so we decided to perform another miniprep to get some more
- Used DB3.1 transformed bacteria from shaker(measured spec 0.794)
- Miniprep
- potential issue our microcentrifuge only spins @ 10,000 not required 12,000
- Binding DNA
- did both optional steps preheated TE + washed w/ W10
- measured UV absorbance = 4.2ng/ul
- in all there is ~ 74 ul of TE should indicated ~370 ng of plasmid.
- Digest
- NOTE: plasmid length w/e biobric is 3189 bp biobrick length is 3255bp this can explain lack of 2 clear bands on gel after ligation.
- nevertheless, we will perform another Gel to make sure we can properly run ladder to determine fragment sizes
- digest measurements: 250ng plasmid all else half except BSA
June 12, 2009
- Ran Gel
- Ladder and other bands were able to be visualized however were still a little wonky
- Suggests for improvement
- increase the glycerol [] in loading dye as there have been problems with diffusion when loading samples as the solution is not falling into the wells
June 15, 2009
- made 1ml and 200ul aliquotes of Kanamycin 10mg/ml stock and stored at -20C
- Recipe: 250ug Kanamycin + 25ml sterile water
- USE : 2x200ul for final working concentration of 20ug/ml
Trouble shooting ladder
- it was noted that the 1x TBE buffer recipe was off. The
