Difference between revisions of "Team:FAU Erlangen/Tour52"

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{{FAU_Erlangen}}
 
{{FAU_Erlangen}}
  
 +
<h4>15/06/30</h4>
  
 +
<b>Primer/rpd3 sequence fragments:</b>
 +
<ul>
 +
<li>centrifugation (5'; 13,000rpm): RPD3 sequences and primer
 +
<li>adjust forward and reverse primer to 100µM (ddH2O) ? working concentration: 5µM (10µl primer + 190µl ddH2O)
 +
<li>adjust RPD3 fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
 +
</ul>
 +
 +
<b>PCR</b>
 +
1x 2,5x
 +
sample:
 +
 +
ddH2O 28.5 µl 71.25 µl
 +
rdp3 fragment 2.1   1 µl   2.5 µl
 +
rdp3 fragment 2.2   1 µl   2.5 µl
 +
dNTP   4 µl 10 µl
 +
Phusion-Polymerase 0.5 µl 1.25 µl
 +
Phusion-Buffer (5x) 10 µl 25 µl
 +
Primer rpd3_fw (5µM)             2.5 µl 6.25 µl
 +
Primer rpd3_rv (5µM)         2.5 µl 6.25 µl
 +
 +
negative control:
 +
 +
ddH2O 30.5 µl 76.25 µl
 +
dNTP   4 µl 10 µl
 +
Phusion-Polymerase 0.5 µl 1.25 µl
 +
Phusion-Buffer (5x) 10 µl 25 µl
 +
Primer rpd3_fw (5µM)         2.5 µl 6.25 µl
 +
Primer rpd3_rv (5µM)         2.5 µl 6.25 µl
 +
 +
PCR Program 1 + 2
 +
 +
<ul>
 +
<li>10µl negativ control-sample + 2µl loading dye
 +
<li>whole PCR tube sample + 8µl loading dye
 +
<li>130 V,  45 min
 +
</ul>
 +
   
 +
<ul>           
 +
<li>cut out both 1718bp fragments
 +
<li>weigh fragments:  fragment PCR programm 1: 0.2g   fragment PCR programm 2: 0.38g
 +
</ul>
 +
 +
<b>DNA fragment extraction (protocol Qiagen gel extraction kit)</b>
 +
<ul>
 +
<li>eluate extracted DNA in 30µl ddH2O
 +
<li>control 1%-Agarosegel: 3µl elution + 2µl loading dye
 +
</ul>
 +
 +
<b>Ligation</b>
 +
<ul>
 +
<li>of rpd3 fragment (PCR program 2) into PCR Blunt/StuI
 +
 +
 +
 +
 +
 +
<h4>15/07/01</h4>
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>Transformation protocol 1
 +
 +
<b>Golden Gate</b>
 +
<ul>
 +
<li>adjust TALE fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
 +
<li>each TAL two samples (? 6 samples)
 +
<li>TALE
 +
</ul>
 +
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)
 +
<ul>
 +
<li>long protocol in ligase buffer
 +
<li>sample:
 +
</ul>
 +
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
 +
 +
<b>Vector pUC19</b>
 +
<ul>
 +
<li>concentration measurment (NanoDrop): ~ 3µg/µl
 +
<li>digestion
 +
</ul>
 +
 +
 +
<b>Golden Gate part 2: control gel</b>
 +
<ul>
 +
<li>10µl TALE sample (1, 2 or 3) + 2µl loading dye
 +
<li>1% Agarose-TAE-Gel
 +
</ul>
 +
           
 +
 +
 +
 +
 +
<h4>15/07/02</h4>
 +
 +
<p>no white colonies</p>
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>Transformation protocol 1 (Repetition rpd3)
 +
 +
<b>Repetition pf T4 Ligation</b>
 +
<ul>
 +
<li>rpd3 fragment PCR programm 1
 +
 +
<b>Golden Gate</b>
 +
<ul>
 +
<li>combine equal samples (6 samples ? 3 samples)
 +
<li>add 0.5µl BsmBI to each sample, incubate 1h, 55°C
 +
<li>add 0.5µl Ligase  to each sample
 +
</ul>
 +
 +
PCR Program 4
 +
 +
<ul>
 +
<li>1% Agarose Gel
 +
</ul>
 +
           
 +
 +
 +
 +
 +
<h4>15/07/06</h4>
 +
 +
<b>Gel extraction</b>
 +
<ul>
 +
<li>TALEs and pUC19 (protocol Qiagen gel extraction kit)
 +
 +
<b>Concentration measurement (NonoDrop)</b>
 +
<ul>
 +
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
 +
 +
<b>Ligation</b>
 +
<ul>
 +
<li>of TALE in pUC19
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>12 test tubes: á 2ml LB medium with Kanamycin
 +
<li>add one white colonie to each tube
 +
<li>incubation: 300rpm, over night, 37°C
 +
</ul>
 +
 +
 +
 +
 +
 +
<h4>15/07/07</h4>
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>Transformation protocol 2 (3x TALE)
 +
 +
 +
 +
 +
 +
<h4>15/07/08</h4>
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of rpd3
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>of TALE
 +
<ul>
 +
<li>6 test tubes: á 2ml LB medium with Kanamycin
 +
<li>add one white colony to each tube (plate1: 2 colonies; plate2: 3colonies; plate3: 1 colony)
 +
<li>incubation: 300rpm, over night, 37°C
 +
</ul>
 +
 +
<b>Gel-electrophorese</b>
 +
<ul>
 +
<li>15µl digestion probe + 3µl loading dye
 +
 +
<b>Sequencing</b>
 +
<ul>
 +
<li>10µl Klon7 + 20µl ddH2O (M13-RP, GATC IJ1672)
 +
 +
 +
 +
 +
 +
<h4>15/07/09</h4>
 +
 +
<b>Golden Gate (2)</b>
 +
<ul>
 +
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
 +
 +
<b>Miniprep (1)</b>
 +
<ul>
 +
<li>culture sample ? protocol „Miniscreens“
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of the mini-prep
 +
<li>with BamHI and SacI
 +
 +
<b>Agarosegel (2)</b>
 +
<ul>
 +
<li>of the Golden Gate
 +
 +
<b>Insert EC culture</b>
 +
<ul>
 +
<li>3 clones, 2ml LB Ampr
 +
 +
<b>Digestion (2)</b>
 +
<ul>
 +
<li>of the Golden Gate
 +
 +
 +
 +
 +
 +
<h4>15/07/10</h4>
 +
 +
<b>Agarosegel (test) (2)</b>
 +
<ul>
 +
<li>over night digestion golden gate insert 2
 +
 +
<b>Ligation: TALE in Vector pUC19_2</b>
 +
<ul>
 +
<li>3.5x Mastermix
 +
 +
<b>Plurification</b>
 +
<ul>
 +
<li>of vector 181 (linearazed) Ampr-Leu
 +
<li>DNA plurification kit
 +
 +
<b>ReTrafo</b>
 +
<ul>
 +
<li>of K801000 iGEM vector Ampr-Ura
 +
 +
<b>Miniprep EC insert</b>
 +
<ul>
 +
<li>Miniscreen protocol, resuspend DNA in 40µl ddH2O
 +
 +
<b>Digestion of EC</b>
 +
<ul>
 +
22.5 µl DNA
 +
3 µl Tango Buffer —> wrong!
 +
0.5 µl EcoRI
 +
4.5µl µl ddH2O
 +
—> 1h @ 37°C
 +
—> inactiviation: 20min @ 65°C
 +
 +
<b>Sequencing</b>
 +
<ul>
 +
<li>10µl clone2 + 20µl ddH2O —> mutations
 +
<li>10µl clone5 + 20µl ddH2O —> mutations
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of Golden Gate (2)
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>prapare 12 tubes with 2.5ml LB + Amp (4 for each insert) for the weekend
 +
 +
 +
 +
 +
 +
<h4>15/07/13</h4>
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of EC Plasmid-DNA + iGEM vector + 181 vector
 +
 +
<b>Miniprep Golden Gate (2)</b>
 +
<ul>
 +
<li>4 samples of each insert
 +
<li>protocoll „Miniscreens“
 +
<li>solve in 40µl ddH2O
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of Golden Gate (2)
 +
 +
<b>Agarose gel</b>
 +
<ul>
 +
<li>of EC + 181 + iGEM vector
 +
<li>+ 5µl Loading Dye
 +
<li>complete digestion (30µl)
 +
       
 +
<b>Plurification</b>
 +
<ul>
 +
<li>EC1 + 2 —> plurification kit
 +
 +
<b>Ligation</b>
 +
<ul>
 +
<li>EC2 ligation into 181 and iGEM
 +
 +
<b>Agarose gel</b>
 +
<ul>
 +
<li>2µl Golden Gate digestion sample + 0.5µl Loading Dye
 +
 +
<b>Sequencing</b>
 +
<ul>
 +
<li>Insert 2_2: 3µl + 17µl ddH2O
 +
 +
<b>Golden Gate (3)</b>
 +
<ul>
 +
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
 +
 +
 +
 +
 +
<h4>15/07/14</h4>
 +
 +
<b>Addition of ligase (3)</b>
 +
<ul>
 +
<li>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
 +
 +
<b>Digestion (3)</b>
 +
<ul>
 +
<li>of Golden Gate-reaction
 +
<li>with BamHI and SacI
 +
<li>Inactivation of the enzymes for 20 min at 80°C
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of the ligation
 +
 +
<b>Agarose gel</b>
 +
<ul>
 +
<li>of the digested Golden Gate reaction
 +
<li>1/5 of the digestion with 1.5 µl Loading Dye 
 +
 +
<b>Sequencing</b>
 +
<ul>
 +
<li>Clone 10 + Clone 11 of rpd3
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>one white colony of the ReTrafo of the iGEM vector in 20ml LB-medium
 +
 +
<b>Ligation (3)</b>
 +
<ul>
 +
<li>of the Golden Gate reaction into pUC19
 +
 +
 +
 +
 +
<h4>15/07/15</h4>
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of the Golden Gate ligation
 +
 +
<b>Mixi-Prep</b>
 +
<ul>
 +
<li>of the iGEM vector
 +
<li>protocol „mixi-prep“
 +
 +
<b>Ligation</b>
 +
<ul>
 +
<li>of EC2-fragment into 181-vector
 +
<li>because there were no white colonies last time, we do a new ligation
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>of 6 clones of EC2 + iGEM-vector
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of the rpd3-ligation
 +
 +
 +
 +
 +
<h4>15/07/16</h4>
 +
 +
<b>Mini-Prep</b>
 +
<ul>
 +
<li>of EC + iGEM-vector
 +
<li>protocol „miniscreens“
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of EC2 + 181-vector ligation (of 15/07/07)
 +
<li>and Golden Gate ligation (of 15/07/14)
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of the mini-prep
 +
 +
<b>Plating</b>
 +
<ul>
 +
<li>of the transformation onto LB-plates containing ampicillin, XGal and IPTG
 +
 +
<b>Agarose gel</b>
 +
<ul>
 +
<li>of the digestion
 +
<li>add 4µl of Loading Dye
 +
 +
         
 +
 +
 +
<h4>15/07/17</h4>
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of the Golden Gate reaction (of 15/07/13), because no colonies grew
 +
<li>first incubate 1h at 37°C with only SacI
 +
<li>then add BamHI and incubate for another hour
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of the 181-vector, because there were only blue colonies on the plates
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>of rpd3 —> 12 clones
 +
<li>and of 2 blue clones of EC + 181 to test them
 +
 +
<b>Ligation</b>
 +
<ul>
 +
<li>of Golden Gate into pUC19
 +
<li>of EC into 181-vector
 +
 +
 +
 +
 +
<h4>15/07/20</h4>
 +
 +
<b>Mini-prep</b>
 +
<ul>
 +
<li>of the fluid cultures from 15/07/17
 +
<li>protocol „miniscreens“
 +
 +
<b>Transformation</b>
 +
<ul>
 +
<li>of the ligations from 15/07/17
 +
 +
<b>Digestion</b>
 +
<ul>
 +
<li>of the mini-preps
 +
 +
<b>Agarose gel</b>
 +
<ul>
 +
<li>of the digestion
 +
<li>rpd3: 15µl with 3µl loading dye
 +
<li>EC/181: 25µl with 5µl loading dye
 +
 +
<b>Sequencing</b>
 +
<ul>
 +
<li>of 2 rpd3 clones
 +
 +
 +
 +
 +
<h4>15/07/22</h4>
 +
 +
<b>Fluid culture</b>
 +
<ul>
 +
<li>of 4 clones for Golden Gate
 +
<li>and 4 clones of EC + 181
 +
 +
 +
 +
 +
<h4>15/07/23</h4>
 +
 +
<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
 
{{FAU_Erlangen_footer}}
 
{{FAU_Erlangen_footer}}

Revision as of 20:21, 18 September 2015

SCATTER
iGEM logo
Introduction
Planning
Execution
Results
Future

15/06/30

Primer/rpd3 sequence fragments:

  • centrifugation (5'; 13,000rpm): RPD3 sequences and primer
  • adjust forward and reverse primer to 100µM (ddH2O) ? working concentration: 5µM (10µl primer + 190µl ddH2O)
  • adjust RPD3 fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing

PCR 1x 2,5x sample:

ddH2O 28.5 µl 71.25 µl rdp3 fragment 2.1 1 µl 2.5 µl rdp3 fragment 2.2 1 µl 2.5 µl dNTP 4 µl 10 µl Phusion-Polymerase 0.5 µl 1.25 µl Phusion-Buffer (5x) 10 µl 25 µl Primer rpd3_fw (5µM) 2.5 µl 6.25 µl Primer rpd3_rv (5µM) 2.5 µl 6.25 µl

negative control:

ddH2O 30.5 µl 76.25 µl dNTP 4 µl 10 µl Phusion-Polymerase 0.5 µl 1.25 µl Phusion-Buffer (5x) 10 µl 25 µl Primer rpd3_fw (5µM) 2.5 µl 6.25 µl Primer rpd3_rv (5µM) 2.5 µl 6.25 µl

PCR Program 1 + 2

  • 10µl negativ control-sample + 2µl loading dye
  • whole PCR tube sample + 8µl loading dye
  • 130 V, 45 min
  • cut out both 1718bp fragments
  • weigh fragments: fragment PCR programm 1: 0.2g fragment PCR programm 2: 0.38g

DNA fragment extraction (protocol Qiagen gel extraction kit)

  • eluate extracted DNA in 30µl ddH2O
  • control 1%-Agarosegel: 3µl elution + 2µl loading dye

Ligation

  • 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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