Team:Brasil-SP/Notebook

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Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Lab Protocols

Competent Cell with Calcium Chloride

  1. Materials

    • Solid LB medium - 1 plate
    • Liquid LB medium
    • 2 250ml centrifuge tubes
    • Centrifuge
    • Cell sample (DH5-alpha in our case)
    • Autoclave
    • Inoculation loop
    • Liquid nitrogen
    • 37°C oven
    • CaCl2:
      • 60mM CaCl2
      • 10mM HEPES
      • 15% glicerol
      • H2O to 100ml

  2. Methods

    3. Day 1

    1. Risk the LB plate with the cell sample (DH5-alpha)
    2. Incubate the plate at 37°C overnight

    Day 2

    1. Sterilize in the Autoclave
      • 100ml of liquid LB medium
      • 100 ml of CaCl2
      • 2 250ml centrifuge tubes
    2. Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight

    Day 3

    1. Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375
    2. Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C
    3. Discard the supernatant and ressuspend the pellet in 10ml of CaCl2 solution
    4. Spin the tubes at 7000rpm/7min/4°C
    5. Discard the supernatant and ressuspend the pellet in 10ml of CaCl2 solution. Incubate 30min on ice
    6. Spin at 7000rpm/7min/4°C
    7. Discard the supernatant and ressuspend the pellet in 2ml of the CaCl2 solution
    8. Distribute the 2ml in 500ul tubes adding 50ul in each.
    9. Freeze the samples in liquid nitrogen
    10. Stock at -80°C

Transformation in Escherichia coli (DH5-alpha)

  1. Materials

    • 1,5ml tube
    • Styrofoam box with ice
    • Agar plate with antibiotic
    • Competent cells
    • Plasmidial DNA
    • Centrifuge
    • Water bath at 42°C
    • Liquid LB medium
    • Shaker

  2. Methods

    1. Briefly spin the competent cells and put then on ice
    2. Add 50ng of plasmidial DNA in a 1,5ml tube
    3. Add the 50ul of competent cell in the same tube
    4. Keep the tube on ice for 25min
    5. Put the tube in a 42° water bath for 2 min
    6. Put the tube on ice for 5 min
    7. Add 200ul of liquid LB
    8. Incubate at 250rpm/37°C/1 hour
    9. Plate the the solution in a Agar plate with the appropriate antibiotic
    10. Incubate the plate at 37°C overnight

Assemblies

Life Inside the LAB

July
Monday Tuesday
    		 Wednesday Thursday Friday Saturday Sunday
    30/06
    • PCR lasR (BBa_C0079) for amplification + RBS addition using primers
    		 01/07
    • Purification of lasR PCR product
    		02/07
    • Transformation of the Biobricks:
      • BBa_143012
      • BBa_K081001
      • BBa_E0840
    • Inoculum of the Biobricks sent by iGEM HQ:
      • BBa_K316037
      • BBa_K316018
      • BBa_K316015
    		 03/07
    • Transformation of the Biobricks sent by the Imperial College Team:
      • BBa_K316016
      • BBa_K143031
    • Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis:
      • BBa_143012
      • BBa_K081001
      • BBa_E0840
    		 04/07 05/07 06/07
    • Inoculum:
      • BBa_143012
      • BBa_K081001
      • BBa_E0840
    07/07
    • Miniprep, Quantification and Restriction Analysis:
      • BBa_143012
      • BBa_K081001
      • BBa_E0840
    • Transformation:
      • BBa_B0015
    		 08/07 09/07
    • Inoculum:
      • BBa_B0015
    		 10/07
    • Miniprep, Quantification and Restriction Analysis:
      • BBa_B0015 (restriction analysis fail, repeat)
    • PCR qteE for amplification + RBS addition using primers
    • Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)
    		 11/07
    • Prepare 40% glycerol solution
    • Transformation:
      • RBS + lasR (BBa_C0079)
    • Purification:
      • RBS + qteE
    		 12/07 13/07
    14/07
    • Assembly:AIII
      • Digestion EXSP
    • Prepare X-gal
    • Prepare more LB medium (solid + liquid)
    		 15/07
    • Assembly:AIII
      • Quantification of digestion EXSP products
    • Replate RBS+lasR
    • Ligation of PCR qteE in pGEM T-easy vector
    • Transformation:
      • BBa_J04450
    • Restriction analysis:
      • BBa_B0015
    		 16/07
    • Tranformation:
      • BBa_K823003
      • RBS+qteE
    • Electrophoresis analysis:
      • BBa_B0015
    		 17/07
    • Glycerol Stock and Miniprep:
      • BBa_J04450
      • RBS+lasR
      • BBa_B0015
    • Digestion:
      • BBa_J04450 (EP)
      • BBa_J04450 (XS)
    • Inoculum:
      • BBa_K823003
    • Transformation
      • BBa_K143055
    		 18/07
    • Glycerol Stock and Miniprep:
      • BBa_J04450
      • BBa_B0015
    • Gel Purification:
      • BBa_J04450 (EP)
      • BBa_J04450 (XS)
    • Restriction analysis:
      • BBa_K823003
    		 19/07 20/07
    21/07
    • Transformation:
      • BBa_316016
    • Inoculum:
      • BBa_K143055
      • RBS+qteE
    • Assembly KI:
      • Digestion EXSP
    		 22/07
    • Inoculum:
      • BBa_K316016
    • Miniprep, Restriction analysis and Digestion:
      • RBS+qteE
    • Assemblies AIII and KI:
      • Gel Purification
      • Ligation
    		 23/07
    • Assemblies AIII and KI:
      • Transformation
    • Prepare more competent cells
    • Prepare LB medium (solid and liquid)
    • Inoculum
      • BBa_K316016
    • Prepare RBS+lasR and RBS+qteE for sequencing
    		 24/07
    • Assemblies AIII and KI:
      • Inoculum
    • Miniprep:
      • BBa_K316016
    		 25/07
    • Assemblies AIII and KI:
      • Glycerol Stock
      • Miniprep
      • Restriction analysis
    		 26/07 27/07
    28/07
    • Assembly AIII:
      • Restriction analysis (+NdeI enzyme)
      • OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that bot the construction and the pSB1C3 vector had similar size. Check out the Lab diary report of the day for more information.
    • Assembly AII AV AVI:
      • Digestion EXSP
    		 29/07
    • >Assembly AII AV AVI:
      • Gel Purification
      • Ligation
    		 30/07 31/07
    • >Assembly AII AV AVI:
      • Inoculum
    • PCR BBa_K143055 for RBS removal
    • OBS: This was one of the parts sent by the Imperial College Team. Because of a comunication failure we assumed that this part was comming without the RBS, so we included a RBS in the parts we were going to use with this Lac promotor :)
    August
    Monday Tuesday Wednesday Thursday Friday Saturday Sunday
    30/07 01/08 02/08 03/08 Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    September
    Monday Tuesday Wednesday Thursday Friday Saturday Sunday
    30/07 01/08 02/08 03/08 Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    October
    Monday Tuesday Wednesday Thursday Friday Saturday Sunday
    30/07 01/08 02/08 03/08 Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points
    First Name Last Name Points Points Points Points Points

    Retrieved from "http://2014.igem.org/Team:Brasil-SP/Notebook"