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of rpd3 fragment (PCR program 2) into PCR Blunt/StuI
15/07/01

Transformation
- Transformation protocol 1 Golden Gate
  - adjust TALE fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
  - each TAL two samples (? 6 samples)
  - TALE
  1. CMV127_1 + CMV127_2 + TALE-CD (= 2416 bp) 2. CMV191_1 + CMV191_2 + TALE-CD (= 2416 bp) 3. GGGT_1 + GGGT_2 + TALE-CD (= 2416 bp)
  - long protocol in ligase buffer
  - sample:
  1x 7x
  T4 ligase buffer (10x) 2 µl 14 µl BSA 2 µl 14 µl T4 Ligase 1 µl 7 µl BsmBI (Esp III) 0.5 µl 3.5 µl 3 TAL fragments each 5.2 µl ddH2O ad 20 µl
  PCR Program 3
  Vector pUC19
  - concentration measurment (NanoDrop): ~ 3µg/µl
  - digestion
  Golden Gate part 2: control gel
  - 10µl TALE sample (1, 2 or 3) + 2µl loading dye
  - 1% Agarose-TAE-Gel
  15/07/02
  
  no white colonies
  
  Transformation
  - Transformation protocol 1 (Repetition rpd3) Repetition pf T4 Ligation
    - rpd3 fragment PCR programm 1 Golden Gate
      - combine equal samples (6 samples ? 3 samples)
      - add 0.5µl BsmBI to each sample, incubate 1h, 55°C
      - add 0.5µl Ligase to each sample
      PCR Program 4
      - 1% Agarose Gel
      15/07/06
      
      Gel extraction
      - TALEs and pUC19 (protocol Qiagen gel extraction kit) Concentration measurement (NonoDrop)
        pUC19 pUC19 TAL1 TAL2 TAL3 0.9 ng/µl 5.4 ng/µl 2.5 ng/µl 2.0 ng/µl 1.4 ng/µl Ligation
        
        of TALE in pUC19 Fluid culture
        
        12 test tubes: á 2ml LB medium with Kanamycin
        add one white colonie to each tube
        incubation: 300rpm, over night, 37°C
        
        15/07/07
        
        Transformation
        
        Transformation protocol 2 (3x TALE)
        15/07/08
        
        Digestion
        
        of rpd3 Fluid culture
        
        of TALE
        
        6 test tubes: á 2ml LB medium with Kanamycin
        add one white colony to each tube (plate1: 2 colonies; plate2: 3colonies; plate3: 1 colony)
        incubation: 300rpm, over night, 37°C
        
        Gel-electrophorese
        
        15µl digestion probe + 3µl loading dye Sequencing
        
        10µl Klon7 + 20µl ddH2O (M13-RP, GATC IJ1672)
        15/07/09
        
        Golden Gate (2)
        
        1x 4x 7x T4 ligase buffer (10x) 2 µl 8 µl 14 µl BSA 2 µl 8 µl 14 µl T4 Ligase 1 µl 4 µl 7 µl BsmBI (Esp III) 0.5 µl 2 µl 3.5 µl TAL fragments each 5.2 µl ddH2O ad 20 µl PCR Program 5 Miniprep (1)
        
        culture sample ? protocol „Miniscreens“ Digestion
        
        of the mini-prep
        with BamHI and SacI Agarosegel (2)
        
        of the Golden Gate Insert EC culture
        
        3 clones, 2ml LB Ampr Digestion (2)
        
        of the Golden Gate
        15/07/10
        
        Agarosegel (test) (2)
        
        over night digestion golden gate insert 2 Ligation: TALE in Vector pUC19_2
        
        3.5x Mastermix Plurification
        
        of vector 181 (linearazed) Ampr-Leu
        DNA plurification kit ReTrafo
        
        of K801000 iGEM vector Ampr-Ura Miniprep EC insert
        
        Miniscreen protocol, resuspend DNA in 40µl ddH2O Digestion of EC
        22.5 µl DNA 3 µl Tango Buffer —> wrong! 0.5 µl EcoRI 4.5µl µl ddH2O —> 1h @ 37°C —> inactiviation: 20min @ 65°C Sequencing
        
        10µl clone2 + 20µl ddH2O —> mutations
        10µl clone5 + 20µl ddH2O —> mutations Transformation
        
        of Golden Gate (2) Fluid culture
        
        prapare 12 tubes with 2.5ml LB + Amp (4 for each insert) for the weekend
        15/07/13
        
        Digestion
        
        of EC Plasmid-DNA + iGEM vector + 181 vector Miniprep Golden Gate (2)
        
        4 samples of each insert
        protocoll „Miniscreens“
        solve in 40µl ddH2O Digestion
        
        of Golden Gate (2) Agarose gel
        
        of EC + 181 + iGEM vector
        + 5µl Loading Dye
        complete digestion (30µl) Plurification
        
        EC1 + 2 —> plurification kit Ligation
        
        EC2 ligation into 181 and iGEM Agarose gel
        
        2µl Golden Gate digestion sample + 0.5µl Loading Dye Sequencing
        
        Insert 2_2: 3µl + 17µl ddH2O Golden Gate (3)
        1x 4x T4 ligase buffer (10x) 2 µl 8 µl BSA 2 µl 8 µl T4 Ligase 1 µl 4 µl BsmBI (Esp III) 0.5 µl 2 µl TAL fragments each 5.2 µl ddH2O ad 20 µl PCR Program 5
        15/07/14
        
        Addition of ligase (3)
        
        because golden gate was not optimal 1x T4 ligase buffer (10x; new) 2 µl T4 Ligase 1 µl BsmBI (Esp III) 0.5 µl Golden Gate reaction; each 10 µl ddH2O 8.5 µl —> 40' @ room temperature Digestion (3)
        
        of Golden Gate-reaction
        with BamHI and SacI
        Inactivation of the enzymes for 20 min at 80°C Transformation
        
        of the ligation Agarose gel
        
        of the digested Golden Gate reaction
        1/5 of the digestion with 1.5 µl Loading Dye Sequencing
        
        Clone 10 + Clone 11 of rpd3 Fluid culture
        
        one white colony of the ReTrafo of the iGEM vector in 20ml LB-medium Ligation (3)
        
        of the Golden Gate reaction into pUC19
        15/07/15
        
        Transformation
        
        of the Golden Gate ligation Mixi-Prep
        
        of the iGEM vector
        protocol „mixi-prep“ Ligation
        
        of EC2-fragment into 181-vector
        because there were no white colonies last time, we do a new ligation Fluid culture
        
        of 6 clones of EC2 + iGEM-vector Transformation
        
        of the rpd3-ligation
        15/07/16
        
        Mini-Prep
        
        of EC + iGEM-vector
        protocol „miniscreens“ Transformation
        
        of EC2 + 181-vector ligation (of 15/07/07)
        and Golden Gate ligation (of 15/07/14) Digestion
        
        of the mini-prep Plating
        
        of the transformation onto LB-plates containing ampicillin, XGal and IPTG Agarose gel
        
        of the digestion
        add 4µl of Loading Dye
        15/07/17
        
        Digestion
        
        of the Golden Gate reaction (of 15/07/13), because no colonies grew
        first incubate 1h at 37°C with only SacI
        then add BamHI and incubate for another hour Digestion
        
        of the 181-vector, because there were only blue colonies on the plates Fluid culture
        
        of rpd3 —> 12 clones
        and of 2 blue clones of EC + 181 to test them Ligation
        
        of Golden Gate into pUC19
        of EC into 181-vector
        15/07/20
        
        Mini-prep
        
        of the fluid cultures from 15/07/17
        protocol „miniscreens“ Transformation
        
        of the ligations from 15/07/17 Digestion
        
        of the mini-preps Agarose gel
        
        of the digestion
        rpd3: 15µl with 3µl loading dye
        EC/181: 25µl with 5µl loading dye Sequencing
        
        of 2 rpd3 clones
        15/07/22
        
        Fluid culture
        
        of 4 clones for Golden Gate
        and 4 clones of EC + 181
        15/07/23
        
        Mini-prep
        
        of the fluid cultures
        protocol „miniscreens“ Digestion
        
        of the mini-preps Agarose Gel
        
        of the digestion
        with 3µl loading dye Overlap-PCR
        
        of rpd3
        PCR program of 15/06/30 Sequencing
        
        of Golden Gate fragment 1 and 3
        15/07/24
        
        Agarose gel
        
        of the overlap-PCR Gel extraction
        
        with Qiagen Gel Extraction Kit Agarose gel
        
        to control the gel extraction <b>Ligation</b>
        
        of rpd3 into PCR/Blunt
        protocol of 15/06/30

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Contents

15/06/30

15/07/01

15/07/02

15/07/06

15/07/07

15/07/08

15/07/09

15/07/10

15/07/13

15/07/14

15/07/15

15/07/16

15/07/17

15/07/20

15/07/22

15/07/23

15/07/24