Team:TorontoMaRSDiscovery/Notebook/June

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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
 +
#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
 +
 +
=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

Revision as of 20:21, 19 October 2009

Home The Team The Project Parts Submitted to the Registry Modeling Bioinformatics Notebook


Contents

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
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