Difference between revisions of "Team:BOKU-Vienna/Notebookdata"

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   <li>PCR plasmid fragments</li>
 
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       <ul>Josef
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         <ul><li>fragment 1: GFP+Primer binding side, fragment 2: GFP + ½ FRT sequence, fragment 3: 1 random sequence
 
         <ul><li>fragment 1: GFP+Primer binding side, fragment 2: GFP + ½ FRT sequence, fragment 3: 1 random sequence
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  <li>Gel/PCR DNA Extraction [PROTOCOL]</li>
 
  <li>Gel/PCR DNA Extraction [PROTOCOL]</li>
 
  <li>NEB Golden Gate Assembly Mix [PROTOCOL]</li>
 
  <li>NEB Golden Gate Assembly Mix [PROTOCOL]</li>
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<li>Miniprep [PROTOCOL]</li>
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  <li>Miniprep [PROTOCOL]</li>
 
   <ul><li>Lisa</li></ul>
 
   <ul><li>Lisa</li></ul>
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Revision as of 12:45, 24 July 2017

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 [PROTOCOL]
  • NEB Golden Gate Assembly Mix [PROTOCOL]
  • Miniprep [PROTOCOL]
    • Lisa