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Team:TorontoMaRSDiscovery/Notebook
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#**Spin down enzymes before using | #**Spin down enzymes before using | ||
#**Overnight digest | #**Overnight digest | ||
| + | |||
| + | =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) | ||
=August 1, 2009= | =August 1, 2009= | ||
Revision as of 02:32, 18 October 2009
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| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook |
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April 27, 2009
- Received from Rosa (SPiT):
- TM0785
- Plasmid containing encapsulin
- Recommend transfect into bacteria and re-sequence
- See email note regarding sequence error
- 0.5 microliters TMG DNA 100 microgram/microliter
- Use 0.4 microliter for 50 microliter PCR reaction =August 1, 2009=
- TM0785
- Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
- The rest of the encapsulin cultures were stocked with 20% glycerol
- 6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
- 16ul of BB5 plasmid was used
- 500ng of plasmid were used for the others
- The digestions were run on a 1.3% agarose gel in TAE (from here on, unless otherwise specified, all gels were 1.3% agarose)
- BB5 was confirmed and all other parts were correct as well
- Overnight ligation of 7+Enc in the PCR machine
August 2, 2009
- Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
- Digested these plasmid samples and ran on gel with negative control (straight from fridge)
- The 3kbp in the digest of 1+2 Sample 5 was not expected
- Comparing the 2 samples of 3+2, the digest of sample 1 did not have a 700bp band, and the undigested sample 1 was about 500bp shorter than the undigested sample 2. We suspect the plasmid in sample 1 did not take up the 3+2 part and somehow recircularized.
- Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates
August 3, 2009
- No colonies were found on 7+Enc plates
August 4, 2009
- Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
- Digested BB4, BB5 and C plasmid, and ran them on a gel
- All bands were of expected sizes.
- Started overnight ligation of 4+5 into C plasmid
August 5, 2009
- Transfected overnight ligation and plated them on C plates
- Plated new C plates (probably meant poured)
- Miniprepped overnight cultures of BB1+2 and K plasmid
August 6, 2009
- Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
- The digests along with negative controls were ran on a gel
- The 1+2 insert was not seen on the gel, probably because it is too small
- The ccdb gene (~600bp) was not seen on the gel
August 7, 2009
- Started overnight cultures of 1+2 sample 1,2,4
- Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
- Plated DH5alpha cells as a control on plain LB plates
- This is thumotoga maritime genomic DNA for purpose of re-cloning
- This is thumotoga maritime genomic DNA for purpose of re-cloning
- Microcentrifuge tubes 1 and 2 placed in -20 freezer
May 15, 2009
- pH buffers received from VWR Mississauga
- pH 4 buffer (red) 500 ml
- pH 7 buffer (yellow) 500 ml
- pH 10 buffer (blue) 500 ml
Above are used for pH/mV Meter calibration
- pH/mV meter calibrated according to manual – recorded in index
- Ethanol solution (70%) made from 85% ethanol
May 19, 2009
- 2L of TE buffer made (10X TE)
- Recipe for 2L from stock solution (10X TE)
- a) 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml
- b) 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml
- c) 988 ml ddH20 x 2 = 1976 ml
- To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl
- 1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used
- Recipe for 2L from stock solution (10X TE)
- 500 ml of 1 M Tris Base made
- mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g
- volume of water used = 500 ml
- 250 ml of 0.5 M EDTA solution was made
- mass of EDTA used = 36.53 g
- observations: EDTA did not dissolve in ddH2O on heat and being stirred
May 21, 2009
- Retrieved autoclaved ddH20, glycerol solution
- Gel Electrophoresis (test run)
- 1% gel: 0.5 g agar mixed with 50 ml of 1X TBE
- 10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead)
- Loading Dye: add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X)
- Running gel: match wells to black side, run at 120 mA
- Visualize Gel in UV
- Turn power on
- Gel in machine face up
- Close door securely
- Turn white light on
- Adjust zoom, contrast, focus from black dial on top of machine
- Turn white light off (turns on UV)
- Press ‘live’ toggle – acq. Should be 0.4 sec.
- Print if desired or save on floppy disk
- Turn power off
- Dispose of gel in proper container
- Close door
May 25, 2009
- Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)
May 26, 2009
- Took overnight cultures from incubator
- Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
- Placed 500 ml flasks into incubator at 37 degrees Celcius
- Grew overnights of DB3.1 from Waterloo (thanks :))
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)
August 1, 2009
- Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
- The rest of the encapsulin cultures were stocked with 20% glycerol
- 6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
- 16ul of BB5 plasmid was used
- 500ng of plasmid were used for the others
- The digestions were run on a 1.3% agarose gel in TAE
- BB5 was confirmed and all other parts were correct as well
- Overnight ligation of 7+Enc in the PCR machine
August 2, 2009
- Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
- Digested these plasmid samples and ran on gel with negative control (straight from fridge)
- The 3kbp in the digest of 1+2 Sample 5 was not expected
- Comparing the 2 samples of 3+2, the digest of sample 1 did not have a 700bp band, and the undigested sample 1 was about 500bp shorter than the undigested sample 2. We suspect the plasmid in sample 1 did not take up the 3+2 part and somehow recircularized.
- Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates
August 3, 2009
- No colonies were found on 7+Enc plates
August 4, 2009
- Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
- Digested BB4, BB5 and C plasmid, and ran them on a gel
- All bands were of expected sizes.
- Started overnight ligation of 4+5 into C plasmid
August 5, 2009
- Transfected overnight ligation and plated them on C plates
- Plated new C plates (probably meant poured)
- Miniprepped overnight cultures of BB1+2 and K plasmid
August 6, 2009
- Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
- The digests along with negative controls were ran on a gel
- The 1+2 insert was not seen on the gel, probably because it is too small
- The ccdb gene (~600bp) was not seen on the gel
August 7, 2009
- Started overnight cultures of 1+2 sample 1,2,4
- Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
- Plated DH5alpha cells as a control on plain LB plates
August 8, 2009
- BB1 transformants showed many colonies
- plates (a plate full of colonies)
- -> increasing heat shock to 90s increased efficiency (Note: 3x DNA used in this transformation)
- Calvin mentioned he does heat shock for 120s
- Calvin also suggested to increase tranformation time, ie. after adding DNA, hold on ice for an hour
- DH-5alpha replates all grew on the plates, demonstrating LB can be re-autoclaved and poured as plates
- there was no difference between transformants directly plated after adding LB+glucose or incubating for an hour
- Miniprepped overnights (1+2) samle 1,2,4, and K
August 10, 2009
- Digested plasmid samples miniprepped on Sat (1,2,4,K) and Enc, C plasmid with EcoR1 and Pst1
- Ran a gel for the digests
- (1+2) Sample 1 appeared to have a ~100bp band
- K plasmid digest did not show the ccdb gene (~700bp) - will start another culture with antibiotics
- Started overnight cultures of (1+2) Sample 1
August 11, 2009
- Digested 4, 5, Enc, C(E+P), C(X+S)
- BB4 did not digest
- 5, C is stored in fridge
- Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
- Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
- Gel extracted C (X+S) after it was CIPed
- Overnight ligation of Enc+C in PCR machine
- Calvin gave us 2 vials of RbCl cells in -80C
- one time use only
- thaw and add DNA
August 12, 2009
- Transformed Enc+C ligations
- Digested BB4 again
- Started overnight ligation of 4+5
- started overnight culture of BB7
- Problem was found in C plates: too soft
- not enough agar?
