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

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 transformations - from Kit Plate 6 InterLab
• 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 Determination of Transformation Efficiency of our DH10B cells:
• Colonies 200µL: 77, 20µL: 26, 100µL: 43

• 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

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

Tuesday, July 25th.

• Minipreparation of BB3_5B and BB3_5F:
• Rebecca
• OneTaq PCR from colonies of BB3_1; BB3_2; BB3_8 and plated onto petri dishes with LB-Hygromycine
• Lisa, Magdalena, Felicia, Mathias, Melanie, Christina
• Some agar plates (BB3_1 + 2 + 8) had too many colonies, so single colonies were plated onto new agar plates
• Felicia, Josef, Magda
• Analytical gel electrophoresis from previous OneTaq PCRs
• Julian, Felicia, Christina
• Transformation (BB1_16, BB1_17 → Hefe guide RNA)
• Julian, Josef

Wednesday, July 26th.

• Colony PCR: BB1_16, BB1_17, BB3_1 + 2 + 8 and plasmid preparation
• Magdalena, Lisa, Valentin, Josef
• Golden Gate with URA3
• Josef, Julian, Christina, Rebecca
• Miniprep:
• BB2_18A; BB2_18D; BB2_18F: Melanie, Felicia
• BB2_18A; BB2_18D; BB2_18F: Valentin, Mathias
• PCR: #38, #39, #40
• Gel/PCR DNA Extraction:
• Felicia
• OD Measurement of preculture from SC288:
• Mathias, Melanie

Thursday, July 27th.

• Colony PCR: BB3_1, 5, 2; BB2_17; BB1_18; CRISPR-PPP
• Magdalena, Felicia, Lisa, Mina
• Miniprep from BB1_16, 17
• Magdalena, Lisa
• PCR #41-43, 31-36 including Gel electrophoresis and gel DNA extraction
• Felicia, Rebecca, Christina, Mathias, Julian
• PCR: #38, #39, #40
• Golden Gate
• BB2_10
• BB2_19 and BB2_10: Josef, Felicia
• Preparing LB-Amp agar plates
• Melanie, Rebecca, Josef

Friday, July 28th.

• Colony PCR
• BB1_20 - BB1_25, BB2_23, BB2_24, BB3_8 + preculture for miniprep in Kanamycin, Hygromycin & Ampicillin Medium
• Magdalena, Felicia, Rebecca
• Preculture for miniprep: BB3_1, BB3_5 (Magdalena, Lisa)
• Miniprep: CRISPR-PPP; BB3_2; BB1_18
• Lisa, Mina
• Sequencing BB3_8
• Christina
• Transformation
• BB2_10,19; BB1_20-25
• LB-Kanamycin plates + agarose gel for gel electrophoresis
• Valentin
• Gel electrophoresis
• CRISPR, BB1_18, BB3_2, BB2_2 and gel DNA extraction
• Christina, Mathias
• BB1_20, 21, 22, 23, 24, BB1_25, BB2_23, BB2_24, BB3_8
• Lisa, Felicia, Rebecca
• BB3_1, BB3_5
• Christina
• Repetition of colony PCR: BB2_23 and BB3_8

Monday, July 31st.

• Colony PCR
• BB2_10; BB2_19; BB2_20; BB3_1; BB3_6; BB3_8 and miniprep pre-cultures in Amp- or Kan- Media
• Lisa, Magda, Mina, Rebecca, Felicia
• Golden Gate Assembly:
• BB2 25-30, CRISPR 2-3 & BB3_2 + Amp, BB1_19
• Josef, Rebecca, Lisa, Christina, Mathias
• Transformation of E. Coli BB2_20 +28+30, BB2_27 + 29 + CRISPR 2 & 3
• Repetition of colony PCR: BB2_19 with new primers (41 fw, 42 rev)
• Magda, Lisa, Mina, Rebecca
• Gel electrophoresis
• Felicia, Josef
• Transformation of yeast
• Lisa, Michael, Rebecca