Team:BOKU-Vienna/Notebook

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Notebook

V

Notebook.


Our Notebook is still under construction. You can find our protocol for the methods here: Protocol

Monday, July 3rd.

  • Team discussion and overview:
    • Christina, Felicia, Josef, Lisa, Magdalena, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian
  • Planning of the procedures including primer design
  • Preparing the growth media and autoclaving
    • LB medium preparation (4L): Mina, Michael
    • Autoclaving: Mina, Rebecca

Tuesday, July 4th.

  • Planning and fundraising:
    • Christina, Felicia, Josef, Lisa, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian
  • Preparation of growth media:
    • 20 plates LB medium + chloramphenicol: Mina, Lisa, Mathias
    • 2 flasks LB agar: Mina, Lisa, Mathias
  • Making cells from straind DH10B chemically competent as a control for the InterLab Study

Wednesday, July 5th.

  • Planning and fundraising:
    • Christina, Felicia, Josef, Lisa, Magdalena, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Julian
  • Aliquoting 1mL LB into Eppendorf tubes
    • Lisa, Josef
  • Transferring Backbones onto agar plates:
    • Julian
  • Interlab challenge - transformation: According to protocol:
      • Lisa, Mathias, Michael, Mina, Rebecca
    • 8 plasmids plated onto agar medium: Positive control (50µL, 200µL), Negative Control (50µL, 200µL), Transformant (20µL, 100µL, 200µL), Test device 1-6 (50µL, 200µL)
    • stored at 35°C

Thursday, July 6th.

  • Team discussion, planning and fundraising
  • Counting colonies of Transformants for the Interlab Challenge:
    • Colonies 200µL: 77, 20µL: 26, 100µL: 43
    Transformants on plate Transformants on plate Transformants on plate
  • Miniprep of 8 plasmids for Golden Gate Standard - according to protocol
    • Magdalena (1), Melanie (2), Valentin (3), Rebeccca (4), Christina (5), Martin (6), Mina (7), Lisa (8)
    • Dilution of plasmids for Golden Gate: Lisa
Dilution sheet for Golden Gate Assembly

Sample Volume of AD - RO Water [µL] Volume of plasmid/fragment [µL]
BB1-C-23-Spy-Cas9(NLS)-49 71,6 28,4
BB1-T-34-sce-cyc-50 83,0 17,0
BB1-P-12-syr-pNS-J23101-25 85,1 14,9
BB1-P-12-sce-tdh3-45 82,7 17,3
BB1-T-34-eco-B1001-29 80,4 19,6
BB1-P-12-syn-pm-J23105-23 83,9 16,1
BB1-C-23-syn-GFP-30 82,9 17,1
BB1-P-12-sce-tef1-46 78,0 22,0

Friday, July 7th.

  • Team discussion, Planning and Fundraising
  • Interlab Challenge: Fluorescein fluorescence standard curve
    • Christina

Monday, July 10th.

  • InterLab Study:
    • Christina, Felicia
  • Dilution of several primers 1:10 (AD-RO Water µL45 , Primer µL5)
    • Magdalena
  • PCR Gel Preparation
    • Mathias

Tuesday, July 11th.

  • Planning & Fundraising
  • Golden Gate Assembly:
    • Flpe (BB1), GFP (BB2) → Transformation (according to protocol) → Plating onto Agar (After Transformation): Selection with Ampicillin (bb1), Selection with Kanamycin(bb2), stored at 37°C over night
  • Dilution of several primer
      • Abbreviation Primer
      1_fw pBAD_fw_FS_1
      2_fw RT_fw_FS2_BpiI
      2_rev RT_rev_RBS_FS_C_BpiI
      3_fw FLPe_fw_FS_C_BpiI
      3_rev FLPe_rev_FS_3_BpiI
      4_fw RepA101ts_fw_FS_23_BsaI
      4_rev RepA101ts_rev_FS_3_BsaI
      5_fw AmpR_Intron_Fw_ohne_FRT_FS_2_BsaI
      5_rev AmpR_Intron_rev_ohne_FRT_FS_3_BsaI
      6_fw KanR_CripsR_fw_FS_B_BsaI
      6_rev KanR_CrispR_rev_Fs_E_BsaI
      7_fw AmpR_Intron_Fw_FRT_FS_2_BsaI
      7_rev AmpR_Intron_rev_FRT_FS_3_BsaI
  • PCR plasmid fragments
    • Josef
    • fragment 1: GFP+Primer binding side, fragment 2: GFP + ½ FRT sequence, fragment 3: 1 random sequence
  • Gel electrophoresis

Wednesday, July 12th.

  • Gel PCR for Kanamycin casette
    • Mina, Magda
  • Restriction digest
  • Gel/PCR DNA Extraction
  • NEB Golden Gate Assembly Mix
  • Miniprep
    • Lisa
  • InterLab Study
    • Felicia, Christina
  • PCR and gel electrophoresis
    • Michael

Thursday, July 13th.

  • Miniprep: Flpe + GFP BB2, control digest
    • Lisa, Mina, Valentin
  • Minis onto LB Kanamycin medium
    • Rebecca
  • Control digest - GFP
    • Mina, Lisa
  • Gel electrophoresis
    • Felicia, Mathias
  • Golden Gate Assembly:
    • Joseph, Felicia, Mina, Christina, Rebecca

Friday, July 14th.

  • Transformation from GG-clones in competent cells using LB medium (with Kan or Amp)
    • Christina, Joseph, Felicia, Melanie, Rebecca
  • Golden Gate Assembly:
    • stored at 37°C
    • Joseph, Felicia, Mina, Christina, Rebecca, Lisa, Mathias, Valentin
  • Gel electrophoresis
    • Felicia, Valentin
  • Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit
    • Felicia, Christina, Valentin, Lisa
  • Concentration measurement of purified DNA
    • Julian

Monday, July 17th.

  • Miniprep
    • control digest
    • BB1_10, BB1_11, BB1_12, BB2_2, BB2_3, BB2_4, BB2_5:
    • Gel electrophoresis
    • Mathias, Mina, Magda, Melanie, Lisa, Rebecca
  • Mini: p5IM5 30°C, Kryo
    • Rebecca
    • Control Transformation
  • PCR
    • Golden Gate: BB2_6 (Rep A, BB2_7: Cas 9, BB2_8: RT, Flpe, BB2_8 → RT, Flpe coli, BB2_1+BB2_5) + Trafo (on LB AMP)
  • Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit
  • Transformation of Golden Gate products:
    • Michi, Julian, Christina, Felicia
  • Kryo for Sequencing
    • Kryo: tube with Glycerine + sample

Tuesday, July 18th.

  • Load gel for gel electrophoresis
    • Melanie
  • Media preparation
    • Melanie, Valentin
  • Colony PCR (11 colonies)
    • Magdalena, Felicia, Christina, Mina
  • Preparation for Sequencing (cryo)
    • Christina
  • more colony PCR
    • Felicia, Christina, Valentin, Rebecca, Lisa
  • PCR Reaction with ThermoCycler

Wednesday, July 19th.

  • PCR gel elektrophoresis
    • Melanie, Christina, Felicia, Magdalena
  • Media preparation (Agar)
    • Lisa, Rebecca
  • Miniprep
    • Christina, Felicia, Valentin, Rebecca
  • Preparation for Sequencing
    • Christina, Melanie
  • Golden Gate
    • Mathias, Felicia, Lisa, Rebecca
  • Transformation for Golden Gate
    • Magdalena, Lisa, Mina, Rebecca, Mathias, Julian, Michael

Thursday, July 20th.

  • Colony PCR:
    • 9 samples: BB2_1 A, B, C, D, E, F und BB2_11 A, B, C
    • Lisa, Christina, Felicia, Rebecca, Lisa, Mina
  • Golden Gate repetition (CRISPR: 5alpha, 10B):
    • Felicia
  • DH10B transformation of Golden Gate products (5alpha, 10B):
    • Christina, Felicia
  • PCR #5
    • Christina
  • Gel electrophoresis
    • with Colony PCR products, Golden Gate products, PCR #5
    • Felicia, Christina
  • DNA purification of PCR #6
    • Christina
  • Aliquoting Master Mix
    • Valentin
  • Golden Gate (GFP, BB2_16)
    • Valentin, Mathias, Rebecca
  • Plating repA colony onto agar
    • Lisa, Valentin
  • Transformation
    • Michael, Julian, Felicia, Christina

Friday, July 21st.

  • Minipreparation:
    • BB2_11A: Christina
  • BB3_5: Yeast in LB Hygromycin
  • Colony PCR
    • Rebecca, Felicia, Melanie, Christina, Mathias
  • Golden Gate PCR - CRISPR
    • Christina, Magdalena, Mathias

Monday, July 24th.

  • Colony PCR: CRISPR (4x), BB3_5 (6x)
    • Lisa, Valentin
  • PCR: yeast sgRNA URA3, yeast sgRNA LEV2
    • Mathias, Christina, Melanie
  • Golden Gate
    • BB3_2, BB3_1, BB3_3, BB3_4, BB3_6, BB2_18, BB3_8
    • Christina, Mathias
  • Golden Gate
    • BB1_16, BB1_17
    • Mina, Felicia