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| | {{FAU_Erlangen}} | | {{FAU_Erlangen}} |
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| − | <h4>15/06/30</h4>
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| − | <b>Primer/rpd3 sequence fragments:</b>
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| − | <ul>
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| − | <li>centrifugation (5'; 13,000rpm): RPD3 sequences and primer
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| − | <li>adjust forward and reverse primer to 100µM (ddH2O) ? working concentration: 5µM (10µl primer + 190µl ddH2O)
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| − | <li>adjust RPD3 fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
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| − | </ul>
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| − |
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| − | <b>PCR</b>
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| − | <ul>
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| − | <li>ddH2O: 28.5 µl
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| − | <li>rdp3 fragment 2.1: 1 µl
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| − | <li>rdp3 fragment 2.2: 1 µl
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| − | <li>dNTP: 4 µl
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| − | <li>Phusion-Polymerase: 0.5 µl
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| − | <li>Phusion-Buffer (5x): 10 µl
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| − | <li>Primer rpd3_fw (5µM): 2.5 µl
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| − | <li>Primer rpd3_rv (5µM): 2.5 µl
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| − | </ul>
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| − |
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| − | <p>negative control:</p>
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| − | <ul>
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| − | <li>ddH2O: 30.5 µl
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| − | <li>dNTP: 4 µl
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| − | <li>Phusion-Polymerase: 0.5 µl
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| − | <li>Phusion-Buffer (5x): 10 µl
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| − | <li>Primer rpd3_fw (5µM): 2.5 µl
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| − | <li>Primer rpd3_rv (5µM): 2.5 µl
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| − | <li>PCR Program 1 + 2
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| − | <br>
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| − | <li>10µl negativ control-sample + 2µl loading dye
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| − | <li>whole PCR tube sample + 8µl loading dye
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| − | <li>130 V, 45 min
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| − | <br>
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| − | <li>cut out both 1718bp fragments
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| − | <li>weigh fragments: fragment PCR programm 1: 0.2g fragment PCR programm 2: 0.38g
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| − | </ul>
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| − |
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| − | <b>DNA fragment extraction (protocol Qiagen gel extraction kit)</b>
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| − | <ul>
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| − | <li>eluate extracted DNA in 30µl ddH2O
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| − | <li>control 1%-Agarosegel: 3µl elution + 2µl loading dye
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| − | </ul>
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| − |
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| − | <b>Ligation</b>
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| − | <ul>
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| − | <li>of rpd3 fragment (PCR program 2) into PCR Blunt/StuI
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| − | </ul>
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/01</h4>
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>Transformation protocol 1
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| − |
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| − | <b>Golden Gate</b>
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| − | <ul>
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| − | <li>adjust TALE fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
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| − | <li>each TAL two samples (? 6 samples)
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| − | <li>TALE
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| − | </ul>
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| − | 1. CMV127_1 + CMV127_2 + TALE-CD (= 2416 bp)
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| − | 2. CMV191_1 + CMV191_2 + TALE-CD (= 2416 bp)
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| − | 3. GGGT_1 + GGGT_2 + TALE-CD (= 2416 bp)
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| − | <ul>
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| − | <li>long protocol in ligase buffer
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| − | <li>sample:
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| − | </ul>
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| − | 1x 7x
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| − |
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| − | T4 ligase buffer (10x) 2 µl 14 µl
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| − | BSA 2 µl 14 µl
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| − | T4 Ligase 1 µl 7 µl
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| − | BsmBI (Esp III) 0.5 µl 3.5 µl
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| − | 3 TAL fragments each 5.2 µl
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| − | ddH2O ad 20 µl
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| − |
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| − | PCR Program 3
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| − |
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| − | <b>Vector pUC19</b>
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| − | <ul>
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| − | <li>concentration measurment (NanoDrop): ~ 3µg/µl
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| − | <li>digestion
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| − | </ul>
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| − |
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| − |
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| − | <b>Golden Gate part 2: control gel</b>
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| − | <ul>
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| − | <li>10µl TALE sample (1, 2 or 3) + 2µl loading dye
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| − | <li>1% Agarose-TAE-Gel
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| − | </ul>
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/02</h4>
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| − |
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| − | <p>no white colonies</p>
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>Transformation protocol 1 (Repetition rpd3)
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| − |
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| − | <b>Repetition pf T4 Ligation</b>
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| − | <ul>
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| − | <li>rpd3 fragment PCR programm 1
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| − |
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| − | <b>Golden Gate</b>
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| − | <ul>
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| − | <li>combine equal samples (6 samples ? 3 samples)
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| − | <li>add 0.5µl BsmBI to each sample, incubate 1h, 55°C
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| − | <li>add 0.5µl Ligase to each sample
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| − | </ul>
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| − |
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| − | PCR Program 4
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| − |
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| − | <ul>
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| − | <li>1% Agarose Gel
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| − | </ul>
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/06</h4>
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| − |
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| − | <b>Gel extraction</b>
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| − | <ul>
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| − | <li>TALEs and pUC19 (protocol Qiagen gel extraction kit)
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| − |
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| − | <b>Concentration measurement (NonoDrop)</b>
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| − | <ul>
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| − | pUC19 pUC19 TAL1 TAL2 TAL3
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| − | 0.9 ng/µl 5.4 ng/µl 2.5 ng/µl 2.0 ng/µl 1.4 ng/µl
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| − |
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| − | <b>Ligation</b>
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| − | <ul>
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| − | <li>of TALE in pUC19
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>12 test tubes: á 2ml LB medium with Kanamycin
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| − | <li>add one white colonie to each tube
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| − | <li>incubation: 300rpm, over night, 37°C
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| − | </ul>
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/07</h4>
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>Transformation protocol 2 (3x TALE)
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/08</h4>
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of rpd3
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>of TALE
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| − | <ul>
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| − | <li>6 test tubes: á 2ml LB medium with Kanamycin
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| − | <li>add one white colony to each tube (plate1: 2 colonies; plate2: 3colonies; plate3: 1 colony)
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| − | <li>incubation: 300rpm, over night, 37°C
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| − | </ul>
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| − |
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| − | <b>Gel-electrophorese</b>
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| − | <ul>
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| − | <li>15µl digestion probe + 3µl loading dye
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| − |
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| − | <b>Sequencing</b>
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| − | <ul>
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| − | <li>10µl Klon7 + 20µl ddH2O (M13-RP, GATC IJ1672)
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/09</h4>
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| − |
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| − | <b>Golden Gate (2)</b>
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| − | <ul>
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| − | 1x 4x 7x
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| − | T4 ligase buffer (10x) 2 µl 8 µl 14 µl
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| − | BSA 2 µl 8 µl 14 µl
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| − | T4 Ligase 1 µl 4 µl 7 µl
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| − | BsmBI (Esp III) 0.5 µl 2 µl 3.5 µl
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| − | TAL fragments each 5.2 µl
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| − | ddH2O ad 20 µl
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| − |
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| − |
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| − | PCR Program 5
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| − |
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| − | <b>Miniprep (1)</b>
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| − | <ul>
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| − | <li>culture sample ? protocol „Miniscreens“
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of the mini-prep
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| − | <li>with BamHI and SacI
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| − |
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| − | <b>Agarosegel (2)</b>
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| − | <ul>
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| − | <li>of the Golden Gate
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| − |
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| − | <b>Insert EC culture</b>
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| − | <ul>
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| − | <li>3 clones, 2ml LB Ampr
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| − |
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| − | <b>Digestion (2)</b>
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| − | <ul>
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| − | <li>of the Golden Gate
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/10</h4>
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| − |
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| − | <b>Agarosegel (test) (2)</b>
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| − | <ul>
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| − | <li>over night digestion golden gate insert 2
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| − |
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| − | <b>Ligation: TALE in Vector pUC19_2</b>
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| − | <ul>
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| − | <li>3.5x Mastermix
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| − |
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| − | <b>Plurification</b>
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| − | <ul>
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| − | <li>of vector 181 (linearazed) Ampr-Leu
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| − | <li>DNA plurification kit
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| − |
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| − | <b>ReTrafo</b>
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| − | <ul>
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| − | <li>of K801000 iGEM vector Ampr-Ura
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| − |
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| − | <b>Miniprep EC insert</b>
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| − | <ul>
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| − | <li>Miniscreen protocol, resuspend DNA in 40µl ddH2O
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| − |
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| − | <b>Digestion of EC</b>
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| − | <ul>
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| − | 22.5 µl DNA
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| − | 3 µl Tango Buffer —> wrong!
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| − | 0.5 µl EcoRI
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| − | 4.5µl µl ddH2O
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| − | —> 1h @ 37°C
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| − | —> inactiviation: 20min @ 65°C
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| − |
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| − | <b>Sequencing</b>
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| − | <ul>
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| − | <li>10µl clone2 + 20µl ddH2O —> mutations
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| − | <li>10µl clone5 + 20µl ddH2O —> mutations
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of Golden Gate (2)
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>prapare 12 tubes with 2.5ml LB + Amp (4 for each insert) for the weekend
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| − |
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/13</h4>
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of EC Plasmid-DNA + iGEM vector + 181 vector
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| − |
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| − | <b>Miniprep Golden Gate (2)</b>
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| − | <ul>
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| − | <li>4 samples of each insert
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| − | <li>protocoll „Miniscreens“
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| − | <li>solve in 40µl ddH2O
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of Golden Gate (2)
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| − |
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| − | <b>Agarose gel</b>
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| − | <ul>
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| − | <li>of EC + 181 + iGEM vector
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| − | <li>+ 5µl Loading Dye
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| − | <li>complete digestion (30µl)
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| − |
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| − | <b>Plurification</b>
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| − | <ul>
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| − | <li>EC1 + 2 —> plurification kit
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| − |
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| − | <b>Ligation</b>
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| − | <ul>
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| − | <li>EC2 ligation into 181 and iGEM
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| − |
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| − | <b>Agarose gel</b>
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| − | <ul>
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| − | <li>2µl Golden Gate digestion sample + 0.5µl Loading Dye
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| − |
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| − | <b>Sequencing</b>
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| − | <ul>
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| − | <li>Insert 2_2: 3µl + 17µl ddH2O
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| − |
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| − | <b>Golden Gate (3)</b>
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| − | <ul>
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| − | 1x 4x
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| − | T4 ligase buffer (10x) 2 µl 8 µl
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| − | BSA 2 µl 8 µl
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| − | T4 Ligase 1 µl 4 µl
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| − | BsmBI (Esp III) 0.5 µl 2 µl
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| − | TAL fragments each 5.2 µl
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| − | ddH2O ad 20 µl
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| − |
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| − | PCR Program 5
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/14</h4>
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| − |
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| − | <b>Addition of ligase (3)</b>
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| − | <ul>
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| − | <li>because golden gate was not optimal
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| − | 1x
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| − | T4 ligase buffer (10x; new) 2 µl
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| − | T4 Ligase 1 µl
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| − | BsmBI (Esp III) 0.5 µl
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| − | Golden Gate reaction; each 10 µl
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| − | ddH2O 8.5 µl
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| − | —> 40' @ room temperature
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| − |
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| − | <b>Digestion (3)</b>
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| − | <ul>
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| − | <li>of Golden Gate-reaction
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| − | <li>with BamHI and SacI
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| − | <li>Inactivation of the enzymes for 20 min at 80°C
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of the ligation
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| − |
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| − | <b>Agarose gel</b>
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| − | <ul>
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| − | <li>of the digested Golden Gate reaction
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| − | <li>1/5 of the digestion with 1.5 µl Loading Dye
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| − |
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| − | <b>Sequencing</b>
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| − | <ul>
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| − | <li>Clone 10 + Clone 11 of rpd3
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>one white colony of the ReTrafo of the iGEM vector in 20ml LB-medium
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| − |
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| − | <b>Ligation (3)</b>
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| − | <ul>
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| − | <li>of the Golden Gate reaction into pUC19
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/15</h4>
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of the Golden Gate ligation
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| − |
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| − | <b>Mixi-Prep</b>
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| − | <ul>
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| − | <li>of the iGEM vector
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| − | <li>protocol „mixi-prep“
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| − |
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| − | <b>Ligation</b>
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| − | <ul>
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| − | <li>of EC2-fragment into 181-vector
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| − | <li>because there were no white colonies last time, we do a new ligation
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>of 6 clones of EC2 + iGEM-vector
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of the rpd3-ligation
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| − |
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| − |
| |
| − |
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| − |
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| − | <h4>15/07/16</h4>
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| − |
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| − | <b>Mini-Prep</b>
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| − | <ul>
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| − | <li>of EC + iGEM-vector
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| − | <li>protocol „miniscreens“
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of EC2 + 181-vector ligation (of 15/07/07)
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| − | <li>and Golden Gate ligation (of 15/07/14)
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of the mini-prep
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| − |
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| − | <b>Plating</b>
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| − | <ul>
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| − | <li>of the transformation onto LB-plates containing ampicillin, XGal and IPTG
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| − |
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| − | <b>Agarose gel</b>
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| − | <ul>
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| − | <li>of the digestion
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| − | <li>add 4µl of Loading Dye
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| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h4>15/07/17</h4>
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| − |
| |
| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of the Golden Gate reaction (of 15/07/13), because no colonies grew
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| − | <li>first incubate 1h at 37°C with only SacI
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| − | <li>then add BamHI and incubate for another hour
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| − |
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| − | <b>Digestion</b>
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| − | <ul>
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| − | <li>of the 181-vector, because there were only blue colonies on the plates
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| − |
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| − | <b>Fluid culture</b>
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| − | <ul>
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| − | <li>of rpd3 —> 12 clones
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| − | <li>and of 2 blue clones of EC + 181 to test them
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| − |
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| − | <b>Ligation</b>
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| − | <ul>
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| − | <li>of Golden Gate into pUC19
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| − | <li>of EC into 181-vector
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| − |
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| − |
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| − |
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| − |
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| − | <h4>15/07/20</h4>
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| − |
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| − | <b>Mini-prep</b>
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| − | <ul>
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| − | <li>of the fluid cultures from 15/07/17
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| − | <li>protocol „miniscreens“
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| − |
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| − | <b>Transformation</b>
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| − | <ul>
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| − | <li>of the ligations from 15/07/17
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| − |
| |
| − | <b>Digestion</b>
| |
| − | <ul>
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| − | <li>of the mini-preps
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| − |
| |
| − | <b>Agarose gel</b>
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| − | <ul>
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| − | <li>of the digestion
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| − | <li>rpd3: 15µl with 3µl loading dye
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| − | <li>EC/181: 25µl with 5µl loading dye
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| − |
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| − | <b>Sequencing</b>
| |
| − | <ul>
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| − | <li>of 2 rpd3 clones
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| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h4>15/07/22</h4>
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| − |
| |
| − | <b>Fluid culture</b>
| |
| − | <ul>
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| − | <li>of 4 clones for Golden Gate
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| − | <li>and 4 clones of EC + 181
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| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h4>15/07/23</h4>
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| − |
| |
| − | <b>Mini-prep</b>
| |
| − | <ul>
| |
| − | <li>of the fluid cultures
| |
| − | <li>protocol „miniscreens“
| |
| − |
| |
| − | <b>Digestion</b>
| |
| − | <ul>
| |
| − | <li>of the mini-preps
| |
| − |
| |
| − | <b>Agarose Gel</b>
| |
| − | <ul>
| |
| − | <li>of the digestion
| |
| − | <li>with 3µl loading dye
| |
| − |
| |
| − | <b>Overlap-PCR</b>
| |
| − | <ul>
| |
| − | <li>of rpd3
| |
| − | <li>PCR program of 15/06/30
| |
| − |
| |
| − | <b>Sequencing</b>
| |
| − | <ul>
| |
| − | <li>of Golden Gate fragment 1 and 3
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h4>15/07/24</h4>
| |
| − |
| |
| − | <b>Agarose gel</b>
| |
| − | <ul>
| |
| − | <li>of the overlap-PCR
| |
| − |
| |
| − | <b>Gel extraction</b>
| |
| − | <ul>
| |
| − | <li>with Qiagen Gel Extraction Kit
| |
| − |
| |
| − | <b>Agarose gel
| |
| − | <ul>
| |
| − | <li>to control the gel extraction
| |
| − |
| |
| − | <b>Ligation</b>
| |
| − | <ul>
| |
| − | <li>of rpd3 into PCR/Blunt
| |
| − | <li>protocol of 15/06/30
| |
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