From 2009.igem.org

Revision as of 03:09, 18 October 2009

April 27, 2009

1. 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=
2. Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
3. The rest of the encapsulin cultures were stocked with 20% glycerol
4. 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
5. 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
6. Overnight ligation of 7+Enc in the PCR machine

August 2, 2009

1. Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
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.
3. Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates

August 3, 2009

1. No colonies were found on 7+Enc plates

August 4, 2009

1. Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
2. Digested BB4, BB5 and C plasmid, and ran them on a gel
   • All bands were of expected sizes.
3. Started overnight ligation of 4+5 into C plasmid

August 5, 2009

1. Transfected overnight ligation and plated them on C plates
2. Plated new C plates (probably meant poured)
3. Miniprepped overnight cultures of BB1+2 and K plasmid

August 6, 2009

1. Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
2. 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

1. Started overnight cultures of 1+2 sample 1,2,4
2. Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
3. Plated DH5alpha cells as a control on plain LB plates
   • • This is thumotoga maritime genomic DNA for purpose of re-cloning
       
4. Microcentrifuge tubes 1 and 2 placed in -20 freezer

May 15, 2009

1. 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
2. pH/mV meter calibrated according to manual – recorded in index
3. Ethanol solution (70%) made from 85% ethanol

May 19, 2009

1. 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

2. 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

3. 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

1. Retrieved autoclaved ddH20, glycerol solution
2. 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
3. Visualize Gel in UV
   1. Turn power on
   2. Gel in machine face up
   3. Close door securely
   4. Turn white light on
   5. Adjust zoom, contrast, focus from black dial on top of machine
   6. Turn white light off (turns on UV)
   7. Press ‘live’ toggle – acq. Should be 0.4 sec.
   8. Print if desired or save on floppy disk
   9. Turn power off
   10. Dispose of gel in proper container
   11. Close door

May 25, 2009

1. Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)

May 26, 2009

1. Took overnight cultures from incubator
2. Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
3. Placed 500 ml flasks into incubator at 37 degrees Celcius
4. Grew overnights of DB3.1 from Waterloo (thanks :))

June 3, 2009

1. 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

1. 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

2. Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
3. Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
4. 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)
5. Inverted and put in 37 degree incubator to dry

June 8, 2009

1. 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)
2. Bacterial liquid culture placed in shaker at 10:51 a.m.

June 9, 2009

1. Digested miniprepped gel with EcoRI and SpeI
2. Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
3. DNA Ladder made - 6 microlitres of stock used

June 10, 2009

1. Poured 10 Tet plates following procedure on June 5, 2009
2. 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

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

August 1, 2009

1. Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
2. The rest of the encapsulin cultures were stocked with 20% glycerol
3. 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
4. The digestions were run on a 1.3% agarose gel in TAE
   • BB5 was confirmed and all other parts were correct as well
5. Overnight ligation of 7+Enc in the PCR machine

August 2, 2009

1. Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
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.
3. Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates

August 3, 2009

1. No colonies were found on 7+Enc plates

August 4, 2009

1. Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
2. Digested BB4, BB5 and C plasmid, and ran them on a gel
   • All bands were of expected sizes.
3. Started overnight ligation of 4+5 into C plasmid

August 5, 2009

1. Transfected overnight ligation and plated them on C plates
2. Plated new C plates (probably meant poured)
3. Miniprepped overnight cultures of BB1+2 and K plasmid

August 6, 2009

1. Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
2. 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

1. Started overnight cultures of 1+2 sample 1,2,4
2. Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
3. Plated DH5alpha cells as a control on plain LB plates

August 8, 2009

1. 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
2. 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
3. Miniprepped overnights (1+2) samle 1,2,4, and K

August 10, 2009

1. Digested plasmid samples miniprepped on Sat (1,2,4,K) and Enc, C plasmid with EcoR1 and Pst1
2. 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
3. Started overnight cultures of (1+2) Sample 1

August 11, 2009

1. Digested 4, 5, Enc, C(E+P), C(X+S)
   • BB4 did not digest
   • 5, C is stored in fridge
2. Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
3. Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
4. Gel extracted C (X+S) after it was CIPed
5. Overnight ligation of Enc+C in PCR machine
6. Calvin gave us 2 vials of RbCl cells in -80C
   • one time use only
   • thaw and add DNA

August 12, 2009

1. Transformed Enc+C ligations
2. Digested BB4 again
3. Started overnight ligation of 4+5
4. started overnight culture of BB7
5. Problem was found in C plates: too soft
   • not enough agar?

August 14, 2009

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