Difference between revisions of "Team:FAU Erlangen/Tour32"
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Adrian FAU (Talk | contribs) |
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<div class="accordionTitel"> Yeast transformation with YFP </div> | <div class="accordionTitel"> Yeast transformation with YFP </div> | ||
| − | <div class="pane" style="height: | + | <div class="pane" style="height:16000px;" > |
| − | < | + | <html> |
| − | < | + | <h1> Yeast transformation with YFP </h1> |
| + | |||
| + | <h4>Day 1, 15/06/25:</h4> | ||
| + | |||
| + | <b> | ||
1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of hostlab's strain collection | 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of hostlab's strain collection | ||
| − | </ | + | </b><br> |
<ul> | <ul> | ||
| − | <li> Strains taken from - | + | <li> Strains taken from -80°C freezer were spread out on YPED-Medium plates |
| − | <li> Incubation for 3 days at | + | <li> Incubation for 3 days at 26°C |
</ul> | </ul> | ||
| − | < | + | <b> |
2. Transformation of Vectors YCplac22, YEplac 181 and YIplac204 in DH5α E. coli | 2. Transformation of Vectors YCplac22, YEplac 181 and YIplac204 in DH5α E. coli | ||
| − | </ | + | </b><br> |
<ul> | <ul> | ||
| − | <li> 1 | + | <li> 1 µl DNA Stocksolution was given onto 50µl bacteria suspension |
<li> Incubation on ice for 30 minutes | <li> Incubation on ice for 30 minutes | ||
| − | <li> Heatshock at | + | <li> Heatshock at 42°C for 90 seconds |
<li> Incubation on ice for 2 minutes | <li> Incubation on ice for 2 minutes | ||
| − | <li> Addition of | + | <li> Addition of 500µl SOC-Medium |
| − | <li> Incubation at | + | <li> Incubation at 37°C for 60 minutes |
| − | <li> | + | <li> 100µl of suspension wase plated on agarplates containing ampicilin |
| − | <li> Incubation at | + | <li> Incubation at 37° C over night |
</ul> | </ul> | ||
| − | < | + | <h4> |
Day 2, 15/06/26: | Day 2, 15/06/26: | ||
| − | </ | + | </h4> |
| − | < | + | <b>1. Picking colonies for overnight cultures (ONK)</b><br> |
<ul> | <ul> | ||
<li> Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000) | <li> Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000) | ||
| − | <li> Afterwards incubation overnight at | + | <li> Afterwards incubation overnight at 37°C |
</ul> | </ul> | ||
| − | < | + | <h4> Day 3, 15/06/29:</h4> |
| − | < | + | <b> 1. DNA-preparation via alkaline Lysis </b><br> |
<ul> | <ul> | ||
<li> Experimental procedure according to " Protocol 1: Alkaline Lysis " | <li> Experimental procedure according to " Protocol 1: Alkaline Lysis " | ||
| − | <li> Changes to protocol:< | + | <li> Changes to protocol: |
| − | + | <ul> | |
| − | + | <li> No centrifugation of ONK | |
| + | <li> Two 2ml reaction tubes filled with ONK instead | ||
| + | </ul> | ||
</ul> | </ul> | ||
| − | < | + | <b> 2. Picking of yeast colonies </b><br> |
<ul> | <ul> | ||
<li> Two yeast colonies picked out of YPED<li>Medium plates from Day 1 (see day 1/1.) | <li> Two yeast colonies picked out of YPED<li>Medium plates from Day 1 (see day 1/1.) | ||
| − | <li> Clone 1 named A | + | <ul> |
| − | <li> Clone 2 named B | + | <li> Clone 1 named A |
| + | <li> Clone 2 named B | ||
| + | </ul> | ||
</ul> | </ul> | ||
| − | < | + | <h4> |
Day 4, 15/06/30: | Day 4, 15/06/30: | ||
| − | </ | + | </h4> |
| − | < | + | <b> |
1. Restriction digest of the obtained DNA-Solutions | 1. Restriction digest of the obtained DNA-Solutions | ||
| − | </ | + | </b><br> |
<ul> | <ul> | ||
<li> Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table | <li> Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table | ||
</ul> | </ul> | ||
| − | |||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th>Reagent</th> | + | <th align="left">Reagent</th> |
| − | <th>Volume for one sample</th> | + | <th align="left">Volume for one sample</th> |
| − | <th>Mastermix for four samples </th> | + | <th align="left">Mastermix for four samples </th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>EcoRI</td> | <td>EcoRI</td> | ||
| − | <td>0.5 | + | <td>0.5 µl</td> |
| − | <td>2 | + | <td>2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>EcoRV</td> | <td>EcoRV</td> | ||
| − | <td>0.5 | + | <td>0.5 µl</td> |
| − | <td>2 | + | <td>2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Tango Buffer</td> | <td>Tango Buffer</td> | ||
| − | <td>4 | + | <td>4 µl</td> |
| − | <td>16 | + | <td>16 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td>Add | + | <td>Add 20µl ddH2O</td> |
| − | <td>14 | + | <td>14 µl</td> |
| − | <td>56 | + | <td>56 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <th> Σ </th> | + | <th align="left"> Σ </th> |
| − | <td>19 | + | <td>19 µl</td> |
| − | <td>76 | + | <td>76 µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
<ul> | <ul> | ||
| − | <li> 19 | + | <li> 19 µl out of the mastermix were transferred to three 1.5 ml reaction tubes |
| − | <li> 1 | + | <li> 1 µl YCplac22/YEplac181/YIplac204 solution obtained in day 3/1 were given in one mastermix tube each |
| − | <li> | + | <li> 1µl K80100 solution obtained in 3/1 was added to following reagents: |
</ul> | </ul> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th>Reagent</th> | + | <th align="left">Reagent</th> |
| − | <th>Volume for one sample</th> | + | <th align="left">Volume for one sample</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PstI</td> | <td> PstI</td> | ||
| − | <td>0.5 | + | <td>0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer O </td> | <td> Buffer O </td> | ||
| − | <td> 2 | + | <td> 2 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> Add | + | <td> Add 20µl ddH2O </td> |
| − | <td> 16.5 | + | <td> 16.5 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <th> Σ </th> | + | <th align="left"> Σ </th> |
| − | <td> 19 | + | <td> 19 µl </td> |
</tr> | </tr> | ||
</table> | </table> | ||
<ul> | <ul> | ||
| − | <li> Digests were incubated at | + | <li> Digests were incubated at 37°C for 3 h |
</ul> | </ul> | ||
| − | < | + | <b> 2. Gelelectrophoresis of digested DNA </b><br> |
<ul> | <ul> | ||
<li> Gelectrophoresis was executed according to "protocol 2: Gelelectrophoresis " | <li> Gelectrophoresis was executed according to "protocol 2: Gelelectrophoresis " | ||
| − | <li> 2 | + | <li> 2 µl 6x staining solution were given unto 10 µl digest |
<li> Samples were loaded onto the gel according to following scheme | <li> Samples were loaded onto the gel according to following scheme | ||
<br> | <br> | ||
| − | ⇒ 1kb-DNA-Marker ( | + | ⇒ 1kb-DNA-Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\1kb DNA-marker (6µl) |
<li> Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes | <li> Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes | ||
| − | < | + | </ul> |
| − | |||
| − | + | <h4> Day 5, 15/07/01:</h4> | |
| − | + | <b> 1. Repetition of restriction digest for Ycplac 204 and K801000 digested </b><br> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | < | + | |
<ul> | <ul> | ||
<li>Mastermix created for BBa_K801000 and YIplac204 | <li>Mastermix created for BBa_K801000 and YIplac204 | ||
| − | + | <br> <br> | |
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th>Reagent</th> | + | <th align="left">Reagent</th> |
| − | <th>Volume for one sample< | + | <th align="left">Volume for one sample</th> |
| − | <th>Mastermix for three samples</th> | + | <th align="left">Mastermix for three samples</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Eco RI</td> | <td>Eco RI</td> | ||
| − | <td> 0.5 | + | <td> 0.5 µl</td> |
| − | <td> 1.5 | + | <td> 1.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> EcoRV </td> | <td> EcoRV </td> | ||
| − | <td> 0.5 | + | <td> 0.5 µl</td> |
| − | <td> 1.5 | + | <td> 1.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Tango Buffer</td> | <td>Tango Buffer</td> | ||
| − | <td>4 | + | <td>4 µl</td> |
| − | <td>12 | + | <td>12 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> Add | + | <td> Add 20µl ddH2O </td> |
| − | <td> 13 | + | <td> 13 µl </td> |
| − | <td> 39 | + | <td> 39 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <th> Σ </th> | + | <th align="left"> Σ </th> |
| − | <td> 18 | + | <td> 18 µl </td> |
| − | <td> 54 | + | <td> 54 µl </td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | <br> | |
| − | <li> | + | <li> 2µl of obtained DNA<li>Solution were transfered to 18 µl of mastermix |
| − | <li> Incubation at | + | <li> Incubation at 37°C for 3 hours |
<li> Gelelectrophoresis was conducted according to protocol 2 | <li> Gelelectrophoresis was conducted according to protocol 2 | ||
| − | <li> 2 | + | <li> 2 µl of 6x staining solution were added onto 10 µl of digest |
| − | <li> Samples were loaded in pockets according to following scheme | + | <li> Samples were loaded in pockets according to following scheme <br> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | ⇒ 6µl 1kb-DNA-marker// YIplac204 digested (15/07/01) // K801000 digested(15/07/01)//YCplac22 (15/06/30)// K801000(15/06/30)// YIplac204 (15/06/30)//empty//YEplac181 (15/06/30)// empty // 6µl 1kb-DNA-marker | ||
</ul> | </ul> | ||
| − | < | + | <b> 2. Preparation of S. Cerevisiae tryptophan negative plates </b><br> |
<ul> | <ul> | ||
<li> Added following substances in two 1 liter flasks: | <li> Added following substances in two 1 liter flasks: | ||
| + | |||
<ul> | <ul> | ||
<li> 3.35g Difco yeast nitrogen base 2/o aminoacid | <li> 3.35g Difco yeast nitrogen base 2/o aminoacid | ||
| Line 251: | Line 247: | ||
</ul> | </ul> | ||
| − | < | + | <b> 3. Preparation of S. Cerevisiae full media plates </b><br> |
<ul> | <ul> | ||
| Line 257: | Line 253: | ||
<ul> | <ul> | ||
| − | <li> | + | <li> 5.3g yeast extract</li> |
<li> 11g Bacopepton</li> | <li> 11g Bacopepton</li> | ||
<li> 10g Glucose</li> | <li> 10g Glucose</li> | ||
| Line 265: | Line 261: | ||
</ul> | </ul> | ||
| − | < | + | <b> 4. Overnightculture of YIplac204 positive bacteria </b><br> |
<ul> | <ul> | ||
| − | <li> | + | <li> Two times 5 ml ampicilin medium (80µg/ml) inoculated with 2 ampicilline ressistant bacteria colonies picked of plate from day 1/2. |
| − | <li> Incubation at | + | <li> Incubation at 37°C over night |
</ul> | </ul> | ||
| − | < | + | <h4> Day 6, 15/07/02: </h4> |
| − | < | + | <b> 1. Moulding of Agar-plates </b><br> |
<ul> | <ul> | ||
<li> Added 500ml destilled Water to each Flask of day 5/2. and day 5/3. | <li> Added 500ml destilled Water to each Flask of day 5/2. and day 5/3. | ||
| Line 284: | Line 280: | ||
</ul> | </ul> | ||
| − | < | + | <b> 2. DNA-Preperation of overnight culture (see day 5/4.)</b><br> |
<ul> | <ul> | ||
<li> Conducted analogous to day 3/1. according to protocol 1 | <li> Conducted analogous to day 3/1. according to protocol 1 | ||
</ul> | </ul> | ||
| − | < | + | <b> 3. Digest of obtained DNA </b><br> |
<ul> | <ul> | ||
| − | <li> Mastermix created to digest 2 | + | <li> Mastermix created to digest 2 µl DNA solution <br> <br> |
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> Reagent </th> | + | <th align="left"> Reagent </th> |
| − | <th> Volume for one sample</th> | + | <th align="left"> Volume for one sample</th> |
| − | <th> Mastermix for three samples</th> | + | <th align="left"> Mastermix for three samples</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> EcoRV </td> | <td> EcoRV </td> | ||
| − | <td> 0.5 | + | <td> 0.5 µl</td> |
| − | <td> 1. | + | <td> 1.5µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer R</td> | <td> Buffer R</td> | ||
| − | <td> 2.0 | + | <td> 2.0 µl</td> |
| − | <td> 6 | + | <td> 6 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> Add | + | <td> Add 20µl H2O </td> |
| − | <td> 15. | + | <td> 15.5µl </td> |
| − | <td> 46. | + | <td> 46.5µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <th> Σ </th> | + | <th align="left"> Σ </th> |
| − | <td> | + | <td> 18µl </td> |
| − | <td> | + | <td> 18µl </td> |
</tr> | </tr> | ||
| Line 328: | Line 324: | ||
<br> | <br> | ||
| − | <li> 2 | + | <li> 2 µl DNA-preparation of each dublicat of YIp204 were added onto 18µl mastermix |
| − | <li> Incubated for 3 hours at | + | <li> Incubated for 3 hours at 37°C |
<li> Electrophoresis was conducted according to protocol 2 | <li> Electrophoresis was conducted according to protocol 2 | ||
| − | <li> Added | + | <li> Added 4µl 6x staining buffer to each digest after incubation time |
| − | <li> Added | + | <li> Added 4µl 6x staining buffer to 20µl undigest DNA<li> Preperation |
<li> Loaded gel according to following scheme | <li> Loaded gel according to following scheme | ||
<br> | <br> | ||
| − | &rArr | + | ⇒ 6µl 1kb-DNA-marker\\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested |
<li> DNA-fragments of unknown origin were found | <li> DNA-fragments of unknown origin were found | ||
| − | + | <li> Repitition of experiment needed | |
| − | + | ||
| − | + | ||
| − | + | ||
</ul> | </ul> | ||
| − | < | + | <b> 4. Overnight culture of strains 4196 and K699 for transformation </b><br> |
<ul> | <ul> | ||
<li> 3 ml YEPD Medium was given to each of 4 reaction tubes | <li> 3 ml YEPD Medium was given to each of 4 reaction tubes | ||
<li> Two yeast colonies of 4196 and K669 were picked | <li> Two yeast colonies of 4196 and K669 were picked | ||
| − | <li> Incubation at | + | <li> Incubation at 30°C over night |
</ul> | </ul> | ||
| − | < | + | <b> 5. Restriction digest of Vector DNA for the transformation </b><br> |
| − | <li> Mix to digest 10 | + | <ul> |
| − | + | <li> Mix to digest 10 µl YIplac204 DNA obtained in preparation 3.1 for transformation created | |
| + | <br> <br> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> Reagent </th> | + | <th align="left"> Reagent </th> |
| − | <th> Volume for one sample </th> | + | <th align="left"> Volume for one sample </th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Eco RV </td> | <td> Eco RV </td> | ||
| − | <td>1 | + | <td>1 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer R </td> | <td> Buffer R </td> | ||
| − | <td> 5 | + | <td> 5 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> Add | + | <td> Add 20µl ddH2O </td> |
| − | <td> 34 | + | <td> 34 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <th> Σ </th> | + | <th align="left"> Σ </th> |
| − | < | + | <td> 40 µl </td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | <br> | |
| − | <li> Incubation over night at | + | <li> Incubation over night at 37°C |
</ul> | </ul> | ||
| − | < | + | <b> 6. Overnight culture of YIplac204 </b><br> |
<ul> | <ul> | ||
| − | <li> 5 ml of ampiciline | + | <li> 5 ml of ampiciline-medium (80µg/ml) were inoculated with one colony obtained of experiment day 1/2. |
| − | <li> Incubation at | + | <li> Incubation at 37°C overnight |
</ul> | </ul> | ||
| − | < | + | <h4> Day 7, 15/07/03 </h4> |
| − | < | + | <b> 1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22 and Ylplac204 </b><br> |
<ul> | <ul> | ||
| − | <li> over night culture diluted 1:100 with MQ (10 | + | <li> over night culture diluted 1:100 with MQ (10 µl suspension and 990 µl MQ) |
<li> measurement 0D600: | <li> measurement 0D600: | ||
| − | + | <br> | |
| − | + | <ul> | |
| − | + | <li> 699 A = 0.203 | |
| − | + | <li> 699 B = 0.143 → used | |
| + | <li> 7196 A = 0.12 | ||
| + | <li> 4196 B = 0.139 → used | ||
| + | </ul> | ||
<li> two flasks filled with YEPG next to Bunsenburner | <li> two flasks filled with YEPG next to Bunsenburner | ||
| − | A: 50 ml | + | <ul> |
| − | B: 25 ml → Media (wrong estimate) | + | <li> A: 50 ml |
| + | <li> B: 25 ml → Media (wrong estimate) | ||
| + | </ul> | ||
<li> in flask A 2 ml suspension 699 oD600 = 0.30 | <li> in flask A 2 ml suspension 699 oD600 = 0.30 | ||
<li> in flask B 1 ml suspension 4196 oD600 = 0.293 | <li> in flask B 1 ml suspension 4196 oD600 = 0.293 | ||
| − | <li> 3 h at 30 | + | <li> 3 h at 30 °C and incubated while shaking |
| − | + | ||
</ul> | </ul> | ||
| − | < | + | <b> 2. DNA-Preparation with YIplac204 overnight culture (see day 6/6.)</b><br> |
<ul> | <ul> | ||
<li> Experiment conducted according to protocol 1 | <li> Experiment conducted according to protocol 1 | ||
</ul> | </ul> | ||
| − | < | + | <b> 3. Gelelectrophoresis of overnight digestion and DNA Preparation </b><br> |
<ul> | <ul> | ||
<li> Experiment conducted according to protocol 2 | <li> Experiment conducted according to protocol 2 | ||
| − | <li> | + | <li> Changes to protocol: |
→ 1.2 % agarose gel | → 1.2 % agarose gel | ||
| − | <li> 5 | + | <li> 5 µl of overnight digest added to 1 µl 6x staining buffer |
<li> loaded onto gel according to following scheme | <li> loaded onto gel according to following scheme | ||
| − | ⇒ 6 | + | ⇒ 6 µl 1kb-DNA-marker \\ overnight digest |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <li> After the photo was taken DNA-Preparation from day 7/2. loaded on same gel | |
| + | <li> Scheme : 6 µl 1kb-DNA-marker //overnight digest //empty// marker // Miniprep | ||
</ul> | </ul> | ||
| − | < | + | <b> 4. Precipitation of plasmid-DNA of digested YIplac204 </b><br> |
<ul> | <ul> | ||
| − | <li> 3.5 | + | <li> 3.5 µl 3M NaAC added to DNA solution |
| − | <li> 1.5 | + | <li> 1.5 µl Glycogen (Thermo Scientific) added |
| − | <li> | + | <li> 100µl 95% Ethanol added |
<li> Incubation for 5 minutes at room temperature | <li> Incubation for 5 minutes at room temperature | ||
<li> Centrifugation (fullspeed, 5 min, room temperature) | <li> Centrifugation (fullspeed, 5 min, room temperature) | ||
<li> DNA appears as a small pellet | <li> DNA appears as a small pellet | ||
<li> Supernatant removed | <li> Supernatant removed | ||
| − | <li> Pellet washed in two volumes (240 | + | <li> Pellet washed in two volumes (240 µl) EtOH 70 % |
<li> Dried at room temperature for 30 min | <li> Dried at room temperature for 30 min | ||
| − | <li> DNA solved in 5 | + | <li> DNA solved in 5 µl TE |
</ul> | </ul> | ||
| − | < | + | <b> 5. Transformation of the yeast K699 </b><br> |
<ul> | <ul> | ||
<li> 25 ml yeast culture from the flask given in 50 ml falcon | <li> 25 ml yeast culture from the flask given in 50 ml falcon | ||
| − | <li> oD determined < | + | <li> oD determined |
| − | + | <ul> | |
| + | <li> 699 = 0.76 | ||
| + | <li> 4196 = 0.75 | ||
| + | </ul> | ||
<li> Centrifugation (5 min, 3500 rpm, room temperature) | <li> Centrifugation (5 min, 3500 rpm, room temperature) | ||
<li> Supernatant discarded | <li> Supernatant discarded | ||
| Line 479: | Line 464: | ||
<li> Centrifugation (10 s, 3500 rpm, room temperature) | <li> Centrifugation (10 s, 3500 rpm, room temperature) | ||
<li> Supernatant discarded | <li> Supernatant discarded | ||
| − | <li> Pellet resuspended in 500 | + | <li> Pellet resuspended in 500 µl 100 mM LiAc |
| − | <li> For each transformation 50 | + | <li> For each transformation 50 µl cell suspension (4) transferred in 1.5ml ml reaction tubes |
<li> Centrifugated for 10 s and supernatant discarded | <li> Centrifugated for 10 s and supernatant discarded | ||
<li> All samples of 4196 discarded (too few DNA) | <li> All samples of 4196 discarded (too few DNA) | ||
| Line 486: | Line 471: | ||
<ul> | <ul> | ||
| − | <li> 240 | + | <li> 240 µl 50 % PEG 3350</li> |
| − | <li> 36 | + | <li> 36 µl 1 M LiAc</li> |
| − | <li> 5 | + | <li> 5 µl carrier DNA (10mg/ml)</li> |
| − | <li> 4 | + | <li> 4 µl YEP122 (positive control) / 5 µl YIP204/ 3µl YCplac22/ 5µl ddH2O (negative control)</li> |
| − | + | → 65 µl of ddH2O to YEplac122 → 360 µl <br> | |
| − | 64 | + | → 64 µl of ddH2O to YIplac204 → 360 µl <br> |
| − | 66 | + | → 66 µl ddH2O to YCplac22 → 360 µl <br> |
| − | 64 | + | → 64 µl ddH2O to negative control → 360 µl |
</ul> | </ul> | ||
<li> Sample vortexed until pellet was resuspended | <li> Sample vortexed until pellet was resuspended | ||
| − | <li> | + | <li> Incubation at room temperature (30 °C) |
| − | <li> heat shock for 20 min at 42 | + | <li> heat shock for 20 min at 42 °C |
| − | <li> centrifugated for 10 s, pellet resuspended in 400 | + | <li> centrifugated for 10 s, pellet resuspended in 400 µl H2O |
<ul> | <ul> | ||
| − | <li> YEplac122 200 | + | <li> YEplac122 200 µl plated</li> |
| − | <li> YIplac204 400 | + | <li> YIplac204 400 µl plated</li> |
| − | <li> YCplac22 200 | + | <li> YCplac22 200 µl plated</li> |
| − | <li> negative control 200 | + | <li> negative control 200 µl plated</li> |
</ul> | </ul> | ||
| − | <li> incubated (72 h, 30 | + | <li> incubated (72 h, 30 °C) |
</ul> | </ul> | ||
| − | < | + | <b> Day 8, 15/07/06: </b><br> |
| − | < | + | <h4> 1. Examination of 7/4.</h4> |
<ul> | <ul> | ||
| Line 518: | Line 503: | ||
</ul> | </ul> | ||
| − | < | + | <h4> 2. Resuspension of IDT geneBricks his_spacer adh1 and his_rep_klein </h4> |
<ul> | <ul> | ||
| − | <li> 500 ng in 50 | + | <li> 500 ng in 50 µl TE given (c = 10 ng/ml) |
| − | <li> incubated (50 | + | <li> incubated (50 °C, 20 min) |
<li> vortexed and centrifugated (10 s at fullspeed) | <li> vortexed and centrifugated (10 s at fullspeed) | ||
</ul> | </ul> | ||
| − | < | + | <h4> 3. Restriction digest for ligation of his_spacer adh1 and his_rep_klein </h4> |
<ul> | <ul> | ||
| − | <li> calculations for needed amount of DNA via following formula | + | <li> calculations for needed amount of DNA via following formula: <br> |
| + | <br> | ||
| − | m(plasmid) * lengt (insert)/length(Vector)*5 | + | m(plasmid) * lengt (insert)/length(Vector)*5 → factor 5 is based on experience |
| + | <br><br> | ||
Thus following values were calculated: | Thus following values were calculated: | ||
| − | + | <br> <br> | |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 556: | Line 543: | ||
</tr> | </tr> | ||
<table> | <table> | ||
| − | + | <br> | |
| − | + | ||
| − | + | ||
| − | + | ||
<li> following restriction digests were constructed: | <li> following restriction digests were constructed: | ||
| − | + | <br><br> | |
| − | < | + | <ul> |
<li>insert his_spacer_adh1/ insert His_Rep_klein</li> | <li>insert his_spacer_adh1/ insert His_Rep_klein</li> | ||
| − | + | <br> | |
| + | </u | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> DNA </th> | + | <th align="left"> DNA </th> |
| − | <th> | + | <th align="left"> 40µl </th> |
| − | <th> MM for 3 samples </th> | + | <th align="left"> MM for 3 samples </th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>EcoRI</td> | <td>EcoRI</td> | ||
| − | <td> | + | <td>2µl </td> |
| − | <td> | + | <td>6µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>PstI </td> | <td>PstI </td> | ||
| − | <td> | + | <td> 2µl </td> |
| − | <td> | + | <td> 6µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer 0 </td> | <td> Buffer 0 </td> | ||
| − | <td> | + | <td> 20µl </td> |
| − | <td> | + | <td> 60µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> add | + | <td> add 200µl ddH2O </td> |
| − | <td> 136 | + | <td> 136 µl</td> |
| − | <td> 408 | + | <td> 408 µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| + | <br> <br> | ||
| − | + | <ul> | |
| − | <li> Vector YEplac181/ YCplac22/ YEplac204 (7/3) | + | <li> Vector YEplac181/ YCplac22/ YEplac204 (7/3)</li> |
| + | </ul> | ||
| + | <br> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> DNA </th> | + | <th align="left"> DNA </th> |
| − | <th> 5 | + | <th align="left"> 5 µl</th> |
| − | <th> MM for 4 samples</th> | + | <th align="left"> MM for 4 samples</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> RNAse </td> | <td> RNAse </td> | ||
| − | <td> 1 | + | <td> 1 µl</td> |
| − | <td> 4 | + | <td> 4 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> EcoRI </td> | <td> EcoRI </td> | ||
| − | <td> 1 | + | <td> 1 µl</td> |
| − | <td> 4 | + | <td> 4 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PstI </td> | <td> PstI </td> | ||
| − | <td> 1 | + | <td> 1 µl</td> |
| − | <td> 4 | + | <td> 4 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer O </td> | <td> Buffer O </td> | ||
| − | <td> 5 | + | <td> 5 µl</td> |
| − | <td> 20 | + | <td> 20 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> add | + | <td> add 50µl ddH2O </td> |
| − | <td> 37 | + | <td> 37 µl</td> |
| − | <td> 148 | + | <td> 148 µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | <br> | |
| + | <ul> | ||
<li> Vector K801000 | <li> Vector K801000 | ||
| + | </ul> | ||
| + | <br> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> DNA </th> | + | <th align="left"> DNA </th> |
| − | <th> 40 | + | <th align="left"> 40 µl</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> RNAse </td> | <td> RNAse </td> | ||
| − | <td> 1 | + | <td> 1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> EcoRI </td> | <td> EcoRI </td> | ||
| − | <td> 2 | + | <td> 2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PstI </td> | <td> PstI </td> | ||
| − | <td> 2 | + | <td> 2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> Buffer O </td> | <td> Buffer O </td> | ||
| − | <td> 10 | + | <td> 10 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> add | + | <td> add 100µl ddH2O </td> |
| − | <td> 45 | + | <td> 45 µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</ol> | </ol> | ||
| + | <br> | ||
| − | <li> large volumes were chosen because of the high EDTA | + | <li> large volumes were chosen because of the high EDTA-concentration |
| − | <li> over night incubation at 37 | + | <li> over night incubation at 37 °C |
</ul> | </ul> | ||
| − | < | + | <b> 4. CloneJet (Thermo Scientific) PCR-cloning with his3_rep_klein and his_spacer_adh1</b><br> |
<ul> | <ul> | ||
| − | <li> as a backup the following | + | <li> as a backup the following plasmids were generated using the CloneJET PCR Cloning Kit |
</ul> | </ul> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <td>2 | + | <td>2 µl </td> |
<td>10 x ligase buffer</td> | <td>10 x ligase buffer</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> 2.5 | + | <td> 2.5 µl </td> |
<td> DNA solution </td> | <td> DNA solution </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td>1 | + | <td>1 µl</td> |
<td>pjet-vector</td> | <td>pjet-vector</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td>add 19 | + | <td>add 19 µl ddH2O </td> |
| − | <td> 13. | + | <td> 13.5µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td>1 | + | <td>1 µl </td> |
<td> T4Ligase </td> | <td> T4Ligase </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | ||
<ul> | <ul> | ||
<li> Incubation (ca. 10 min) | <li> Incubation (ca. 10 min) | ||
| Line 704: | Line 696: | ||
</ul> | </ul> | ||
| − | < | + | <b> 5. Transformation</b><br> |
<ul> | <ul> | ||
| − | <li> DH5alpha< | + | <li> DH5alpha <i>E. coli</i> unfreezed |
| − | <li> Each 5 | + | <li> Each 5 µl from Day8 4. given on top of 50 µl competent cells |
| − | <li> As a positive control | + | <li> As a positive control 1µl PBSC<li>Bluescript was given on top of 50 µl E.coli |
| − | <li> As a negative control no changes were applied to 50 | + | <li> As a negative control no changes were applied to 50 µl E. coli |
<li> incubate for about 15 min on ice | <li> incubate for about 15 min on ice | ||
<li> heat shock for 90 s | <li> heat shock for 90 s | ||
<li> 2 min on ice | <li> 2 min on ice | ||
| − | <li> 500 | + | <li> 500 µl SOC medium added |
| − | <li> incubated (45 min, 37 | + | <li> incubated (45 min, 37 °C) |
<li> centrifugation (2 min, 7000 rpm) | <li> centrifugation (2 min, 7000 rpm) | ||
| − | <li> remove 450 | + | <li> remove 450 µl supernatant |
| − | <li> E.coli in remained 100 | + | <li> E.coli in remained 100 µl resuspended |
| − | <li> 100 | + | <li> 100 µl plated on ampiciline plates |
</ul> | </ul> | ||
| − | < | + | <h4> Day 9, 15/07/07 </h4> |
| − | < | + | <b> 1. Analysis of over night digest from Day 8/3. </b><br> |
<ul> | <ul> | ||
| Line 737: | Line 729: | ||
<td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">YEplac181/ YCplac22/YIplac204</span></p> | <td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">YEplac181/ YCplac22/YIplac204</span></p> | ||
</td> | </td> | ||
| − | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">5 | + | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">5 µl</span></p> |
</td> | </td> | ||
| − | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(1 | + | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(1 µl)</span></p> |
</td> | </td> | ||
</tr> | </tr> | ||
| Line 745: | Line 737: | ||
<td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">Bba_K801000</span></p> | <td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">Bba_K801000</span></p> | ||
</td> | </td> | ||
| − | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">10 | + | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">10 µl</span></p> |
</td> | </td> | ||
| − | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(2 | + | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(2 µl)</span></p> |
</td> | </td> | ||
</tr> | </tr> | ||
| Line 753: | Line 745: | ||
<td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">Inserts (his3_rep_klein/ his_spacer_adh1</span></p> | <td width="261" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">Inserts (his3_rep_klein/ his_spacer_adh1</span></p> | ||
</td> | </td> | ||
| − | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">20 | + | <td width="68" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">20 µl</span></p> |
</td> | </td> | ||
| − | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(4 | + | <td width="53" style="border-width : 0px;"><p style=" text-align: left; text-indent: 0px; padding: 0px 0px 0px 0px; margin: 0px 0px 0px 0px;"><span style=" font-size: 10pt; font-family: 'Arial', 'Helvetica', sans-serif; font-style: normal; font-weight: normal; color: #000000; background-color: transparent; text-decoration: none;">(4 µl)</span></p> |
</td> | </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| − | + | <br> | |
| − | + | <ul> | |
<li> Pockets were loaded according to following scheme | <li> Pockets were loaded according to following scheme | ||
<br> | <br> | ||
⇒ 1 kb DNA-marker //YEplac181//YCplac22//YIplac204//K801000//his_spacer_adh1//his3_rep_klein | ⇒ 1 kb DNA-marker //YEplac181//YCplac22//YIplac204//K801000//his_spacer_adh1//his3_rep_klein | ||
</ul> | </ul> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | + | <b> 2. Restriction digestion 204 </b><br> | |
| − | < | + | |
<ul> | <ul> | ||
| − | <li> 40 | + | <li> 40 µl plasmid solution 204 obtained out of Day 7 2. digested |
| − | < | + | </ul> |
| − | <table border=" | + | <br> |
| + | <table border="0" width="75%"> | ||
<tr><!-- Row 1 --> | <tr><!-- Row 1 --> | ||
<td>DNA </td><!-- Col 1 --> | <td>DNA </td><!-- Col 1 --> | ||
| − | <td>40 | + | <td>40 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
<tr><!-- Row 2 --> | <tr><!-- Row 2 --> | ||
<td>EcoRI</td><!-- Col 1 --> | <td>EcoRI</td><!-- Col 1 --> | ||
| − | <td>2 | + | <td>2 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
<tr><!-- Row 3 --> | <tr><!-- Row 3 --> | ||
<td>PstI</td><!-- Col 1 --> | <td>PstI</td><!-- Col 1 --> | ||
| − | <td>2 | + | <td>2 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
<tr><!-- Row 4 --> | <tr><!-- Row 4 --> | ||
<td>Buffer O</td><!-- Col 1 --> | <td>Buffer O</td><!-- Col 1 --> | ||
| − | <td>20 | + | <td>20 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
<tr><!-- Row 5 --> | <tr><!-- Row 5 --> | ||
<td>H2O</td><!-- Col 1 --> | <td>H2O</td><!-- Col 1 --> | ||
| − | <td>136 | + | <td>136 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
<tr><!-- Row 6 --> | <tr><!-- Row 6 --> | ||
<td>Σ</td><!-- Col 1 --> | <td>Σ</td><!-- Col 1 --> | ||
| − | <td>200 | + | <td>200 µl</td><!-- Col 2 --> |
</tr> | </tr> | ||
</table> | </table> | ||
<ul> | <ul> | ||
| − | <li> incubation (2h, 37 | + | <li> incubation (2h, 37 °C) |
</ul> | </ul> | ||
| − | < | + | <b> 3. Gelelectrophoresis for extraction </b><br> |
<ul> | <ul> | ||
<li> Gelelectrophoresis conducted according to protocol 2 | <li> Gelelectrophoresis conducted according to protocol 2 | ||
| − | <li> Middlepocket of gel loaded with 45 | + | <li> Middlepocket of gel loaded with 45 µl YCplac22 Day 8 3.according to following scheme: <br> |
| − | ⇒ | + | ⇒ 6µl 1 kb DNA<li>marker // empty // YCplac22 // empty |
<ul> | <ul> | ||
<li> Changes to protocol: | <li> Changes to protocol: | ||
| Line 818: | Line 806: | ||
</ul> | </ul> | ||
| − | < | + | <b> 4. Gelextraction via Quiagen kit</b><br> |
<ul> | <ul> | ||
<li> Gel fragment weighted: 470 mg | <li> Gel fragment weighted: 470 mg | ||
| − | <li> 3 times the volume QC-buffer added → 1410 | + | <li> 3 times the volume QC-buffer added → 1410 µl QC-buffer added |
| − | <li> 10 min at 50 | + | <li> 10 min at 50 °C gel dissolved |
<li> After 3 min and 7 min for 5-7 s vortexed | <li> After 3 min and 7 min for 5-7 s vortexed | ||
| − | <li> 450 | + | <li> 450 µl isopropanol added |
<li> Sample inverted and shortly vortexed | <li> Sample inverted and shortly vortexed | ||
| − | <li> 800 | + | <li> 800 µl given on speedcolumn with wastetube |
<li> Centrifugation (13300 rpm, 1 min) → flowthrough discarded | <li> Centrifugation (13300 rpm, 1 min) → flowthrough discarded | ||
<li> Step 3 times repeated | <li> Step 3 times repeated | ||
| Line 836: | Line 824: | ||
<li> Flowthrough discarded | <li> Flowthrough discarded | ||
<li> Column transferred on 1.5 ml tube | <li> Column transferred on 1.5 ml tube | ||
| − | <li> 30 | + | <li> 30 µl Elutionbuffer added on column |
<li> Centrifugation (13300 rpm, 1 min) | <li> Centrifugation (13300 rpm, 1 min) | ||
<li> Eluate used for further experiments | <li> Eluate used for further experiments | ||
</ul> | </ul> | ||
| − | < | + | <b> 5. Further cleaning with Quiagen PCR-purification-kit </b><br> |
<ul> | <ul> | ||
| − | <li> 180 | + | <li> 180 µl sample given to 900 µl PB buffer given |
| − | <li> 800 | + | <li> 800 µl Sample given on column |
<li> column centrifugated (13300 rpm, 1 min) | <li> column centrifugated (13300 rpm, 1 min) | ||
| − | <li> remaining 280 | + | <li> remaining 280 µl given on column |
<li> column centrifugated at 13300 rpm | <li> column centrifugated at 13300 rpm | ||
<li> both times flowthrough was discarded | <li> both times flowthrough was discarded | ||
| − | <li> column loaded with 750 | + | <li> column loaded with 750 µl PE |
<li> centrifugation (13300 rpm, 1 min) | <li> centrifugation (13300 rpm, 1 min) | ||
<li> flowthrough discarded | <li> flowthrough discarded | ||
| − | <li> | + | <li> centrifugation (13300 rpm, 1 min) |
| + | <li> flowthrough discarded | ||
| + | <li> column transferred on 1.5 ml Eppi | ||
| + | <li> 30 µl EB (elution buffer) given | ||
| + | <li> centrifugation (13300 rpm, 1 min) | ||
| + | <li> eluate used for further experiments | ||
| + | </ul> | ||
| + | |||
| + | <b> 6. Controlgelelectrophoresis of eluate and digestion of YIplac204 and YCplac22 (day 9/2.) </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Gelelectrophoresis according to protocol 2: Electrophoresis | ||
| + | <li> Gel loaded with samples according to following scheme: | ||
| + | </ul> | ||
| + | |||
| + | ⇒ Standard (6µl) // YCplac22 (3µl) //YIplac204 digested (20µl) // empty // Standard (6µl) // Insert his3_reporter_klein (2.8µl) | ||
| + | <ul> | ||
| + | <li>Changes to protocol: | ||
| + | <ul> | ||
| + | <li> Gelelectrophoresis for 45 min | ||
| + | <li> note: insert was added after 20 min | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <b> 7. Overnight culture of Pjet-transformed E. coli </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 2 colonies on plate 204, Pjet_His_spacer, Pjet_reporter_klein picked | ||
| + | <li> Each were given into 5 ml 80 µg/µl ampiciline medium given | ||
| + | <li> over night incubation at 37 °C | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 10, 15/07/08: </h4> | ||
| + | |||
| + | <b> 1. Ligation of the linearised vector YCplac22 and the his3_rep_klein </b> <br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 3 samples for ligation generated | ||
| + | <ul> | ||
| + | <li> ligation with insert | ||
| + | <ul> | ||
| + | <li> 3 µl vector (YCPlac22) linearized | ||
| + | <li> 14 µl insert (His3_rep_klein/ his_spacer_adh1) linearized | ||
| + | <li> 2 µl ligase buffer | ||
| + | <li> 1 µl T4 Ligase | ||
| + | </ul> | ||
| + | |||
| + | <li> Religation control without insert | ||
| + | <ul> | ||
| + | <li> 3 µl vector (YCPlac22) linearized | ||
| + | <li>14 µl ddH2O | ||
| + | <li> 2 µl ligase buffer | ||
| + | <li> 1 µl T4 Ligase | ||
| + | </ul> | ||
| + | <li> control for complete digestion | ||
| + | <ul> | ||
| + | <li> 3 µl vector (YCPlac22) linearized | ||
| + | <li> 15 µl H2O | ||
| + | <li> 2 µl ligase buffer | ||
| + | <li> ligation incubated for 3 h at room temperature | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Miniprep from overnight culture day 9/7.</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 2 ml over night culture transferred to 2 ml<li>reaction tubes | ||
| + | <li> centrifugation (7000 rpm, 5 min) | ||
| + | <li> supernatant discarded | ||
| + | <li> 2 ml from over night culture transferred in the same reaction tubes as their precursors to increase the amount of bacteria | ||
| + | <li> centrifugation (7000 rpm, 5 min) | ||
| + | <li> supernatant discarded | ||
| + | <li> DNA<li>Preparation conducted as given by Quia<li>Gen Miniprep instruction | ||
| + | <li> Elution in 50 µl elution buffer | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Digestion of Miniprep </b><br> | ||
| + | <ul> | ||
| + | <li>Digest prepared | ||
| + | </ul> | ||
| + | <table> | ||
| + | <table border="0" width="70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <th align="left"></th><!-- Col 1 --> | ||
| + | <th align="left">1 µl DNA from the miniprep(see above)</th><!-- Col 2 --> | ||
| + | <th align="left">MM for 7 samples à 19 µl</th><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>EcoRI </td><!-- Col 1 --> | ||
| + | <td>0.5 µl</td><!-- Col 2 --> | ||
| + | <td>3.5 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>PstI</td><!-- Col 1 --> | ||
| + | <td>0.5 µl </td><!-- Col 2 --> | ||
| + | <td>3.5 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>Buffer O</td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | <td>14 µl </td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>ddH2O</td><!-- Col 1 --> | ||
| + | <td>16 µl </td><!-- Col 2 --> | ||
| + | <td>112 µl </td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ul> | ||
| + | <li> digestion for 3 h at 37 °C | ||
| + | </ul> | ||
| + | |||
| + | <b> 4. Moulding of Chloramphenicol agar plates </b><br> | ||
| + | <ul> | ||
| + | <li> TY-Medium with agar heated | ||
| + | <li> Cooling while mixing | ||
| + | <li> Addition of 50 µl Chloramphenicol (100 mg/ml) | ||
| + | <li> c_end= 10µg/ml | ||
| + | <li> Moulding of plates | ||
| + | </ul> | ||
| + | |||
| + | <b> 5 Preparation of X-gal plates </b><br> | ||
| + | <ul> | ||
| + | <li> 40µl 2% x-gal spread on 6 ampicilin enriched plates | ||
| + | <li> 40µl IPTG spread on the dried plates | ||
| + | <li> 40µl dH2O spread on the dried plates | ||
| + | </ul> | ||
| + | |||
| + | <b> 6 Transformation of Ligation and EYFP (BBa_E2030)</b><br> | ||
| + | <ul> | ||
| + | <li> Each of folowing DNA<li>samples added to 50 µl DH5α on ice | ||
| + | <ul> | ||
| + | <li>5µl ligation from 10.1 | ||
| + | <li>5µl ligase + vectorligation from 10.1 | ||
| + | <li>5µl vector + ligase buffer solution from 10.1 | ||
| + | <li>1µl pBluescript | ||
| + | <li>5µl H2O | ||
| + | <li>2.5µl EYFP (BBa_E2030) | ||
| + | </ul> | ||
| + | |||
| + | <li>Transformation analogous to 8.5 | ||
| + | <li>Transformed <i>E. coli</i> samples spread on ampiciline plates as followed | ||
| + | <ul> | ||
| + | <li> 100 µl ligation | ||
| + | <li> 200 µl ligation | ||
| + | <li> 200 µl ligase + vector | ||
| + | <li> 200 µl pBluescript | ||
| + | <li> 200 µl H2O | ||
| + | </ul> | ||
| + | |||
| + | <li>Remaining transformed bacteria spread on chloramphenicole agar plates | ||
| + | <ul> | ||
| + | <li> 200 µl EYFP (BBa_E2030) | ||
| + | <li> 200 µl H2O | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <b> 7. Gelelectrophoresis of the Digestion from 3. (see above)</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Gelelectrophoresis conducted according to protocol 2 | ||
| + | <li> 20 µl taken from earlier digest (see above) and added to 4µl Staining Solution | ||
| + | <li> Gel loaded according to following scheme: | ||
| + | </ul> | ||
| + | |||
| + | ⇒ his3_rep_klein A // his3_rep_klein B // his_spacer_adh1 A // his_spacer_adh1 B// YIplac204 A //YIplac 204 B// 1kb DNA-marker | ||
| + | |||
| + | <h4>Day 11, 15/07/09</h4> | ||
| + | |||
| + | <b>1. Cultures picked for over night culture </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 12 colonies taken from 100 µl plate (compare Day 10 6.) | ||
| + | <li> inoculation of 2 ml 80 µg/ml ampiciline medium | ||
| + | <li> 2 colonies picked from YFP<li>plate | ||
| + | <li> 5 ml 25 µg/ml Canamycin medium inoculated | ||
| + | <li> over night incubation at 37 °C | ||
| + | </ul> | ||
| + | |||
| + | <h4>Day 12, 15/07/10</h4> | ||
| + | |||
| + | <b> 1. Miniprep of the over night culture from day 11/1. (see above)</b><br> | ||
| + | <ul> | ||
| + | <li> conducted according to protocol 1 | ||
| + | <li> eluted in 30µl 1x TE | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Restriction digestion </b><br> | ||
| + | <ul> | ||
| + | <li> Restriction for all DNA-preparations (14 samples) | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="100%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>DNA Solution</td><!-- Col 1 --> | ||
| + | <td>3 µl </td><!-- Col 2 --> | ||
| + | <td>MM for 15</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>PstI</td><!-- Col 1 --> | ||
| + | <td>0.5 µl</td><!-- Col 2 --> | ||
| + | <td>7.5 µl </td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>EcoRI</td><!-- Col 1 --> | ||
| + | <td>0.5 µl</td><!-- Col 2 --> | ||
| + | <td>7.5 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>Buffer O</td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | <td>30 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | |||
| + | <tr><!-- Row 4 --> | ||
| + | <td>ddH2O</td><!-- Col 1 --> | ||
| + | <td>14 µl</td><!-- Col 2 --> | ||
| + | <td>210 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <ul> | ||
| + | <li> incubation (1.5 h, 37 °C) | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Gelelectrophoresis</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Gelelectrophoresis performed according to protocol 2 | ||
| + | <li> Changes to protocol: | ||
| + | <ul> | ||
| + | <li> Volume for two 1% agarosegels produced 170 ml (100 ml+70 ml) | ||
| + | <li> 13,6 µl Roth Ethidiumbromide added | ||
| + | </ul> | ||
| + | <li> Samples from Day 12 2. (see above) were added to 4 µl staining solution | ||
| + | <li> Gels were loaded as followed: | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <table border="0" width="57.3%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>Gel I:</td><!-- Col 1 --> | ||
| + | <td>1kb DNA-marker// YCPlac22 + insert</td><!-- Col 2 --> | ||
| + | <td> 1 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>2 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>3 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>4 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>5 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>6 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 7 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>7 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 8 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>8 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <br><br> | ||
| + | |||
| + | <table border="0" width="70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>Gel II:</td><!-- Col 1 --> | ||
| + | <td>1kb DNA-marker // YCPlac22 + insert </td><!-- Col 2 --> | ||
| + | <td>9 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>10 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>11 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>12 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>empty//</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 6 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>YFP clone 1 //</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 7 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>YFP clone 2//</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 8 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td></td><!-- Col 2 --> | ||
| + | <td>stamdard//</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <ul> | ||
| + | <li> Results not expected ratio insert vector seems tilted | ||
| + | <li> Over night culture of all samples analogous to Day 11/1. | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <h4> Day 13, 15/07/11 </h4> | ||
| + | |||
| + | <b> 1. Miniprep via Quiagen column </b> <br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 5 ml over night culture from day 12/3. centrifuged (7000 rpm, 5min) in 2 ml Eppis | ||
| + | <li> Quiagen Miniprep analogous to 10/2. performed | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Restriction digestion </b> <br> | ||
| + | <ul> | ||
| + | <li> DNA-Solutions obtained in Day 13/1. (see above) EcoRI PstI digested | ||
| + | <li> Mastermix created | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="100%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <th align="left">single sample</th><!-- Col 1 --> | ||
| + | <th align="left">Mastermix for 13 Samples</th><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td> 1 µl DNA </td><!-- Col 1 --> | ||
| + | <td>-</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>0.5 µl EcoRI</td><!-- Col 1 --> | ||
| + | <td>6.5 µl EcoRI</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>0.5 µl PstI </td><!-- Col 1 --> | ||
| + | <td>6.5 µl PstI</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>2 µl Buffer0</td><!-- Col 1 --> | ||
| + | <td>26 µl Buffer0</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 6 --> | ||
| + | <td> 16 µl ddH2O</td><!-- Col 1 --> | ||
| + | <td>208 µl ddH2O</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ul> | ||
| + | <li> incubation (2 h, 37 °C) | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Gelelectrophoresis </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Gelelectrophoresis conducted according to protocol 2 | ||
| + | <li> 4 µl staining solution added to each of the 12 samples from experiment Day 13/2. (see above) | ||
| + | <li> 24 µl loaded on gel according to following scheme | ||
| + | <ul> | ||
| + | <li> 1kb DNA-marker // | ||
| + | <li> YCPlac 22 + insert 1 // | ||
| + | <li> YCPlac 22 + insert 2 // | ||
| + | <li> YCPlac 22 + insert 3 // | ||
| + | <li> YCPlac 22 + insert 4 // | ||
| + | <li> ... | ||
| + | <li> YCPlac 22 + insert 12 // | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 14, 15/07/14</h4> | ||
| + | |||
| + | <b> 1. Restriction digestion XhoI, Sal I from YCPlac22 + insert10 (day 13/13)</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> restriction digestion conducted | ||
| + | </ul> | ||
| + | <table border="0" width="70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>DNA YCPlac22 + insert10 from day 13</td><!-- Col 1 --> | ||
| + | <td>20 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td> Sal I</td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>XhoI</td><!-- Col 1 --> | ||
| + | <td>4 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>Buffer 0</td><!-- Col 1 --> | ||
| + | <td>10 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>ddH2O</td><!-- Col 1 --> | ||
| + | <td>64 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ul> | ||
| + | <li> incubation (2 h, 37 °C) | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Addition of SalI and XhoI restriction sites via PCR </b><br> | ||
| + | <br> | ||
| + | <table border="0" width="70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <th align= "left">Sample </th><!-- Col 2 --> | ||
| + | <th align= "left" >water control</th><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>H2O </td><!-- Col 1 --> | ||
| + | <td>71 µl </td><!-- Col 2 --> | ||
| + | <td>73 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>5 x buffer </td><!-- Col 1 --> | ||
| + | <td>20 µl </td><!-- Col 2 --> | ||
| + | <td>20 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>template </td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | <td>-</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>Forward Primer </td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | <td>2 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 6 --> | ||
| + | <td>Reverse Primer </td><!-- Col 1 --> | ||
| + | <td>2 µl </td><!-- Col 2 --> | ||
| + | <td>2 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 7 --> | ||
| + | <td>Fusion Polymerase </td><!-- Col 1 --> | ||
| + | <td>1 µl </td><!-- Col 2 --> | ||
| + | <td>1 µl</td><!-- Col 3 --> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th align="left"> Σ </th> | ||
| + | <td> 100 µl </td> | ||
| + | <td> 100 µl </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | <ul> | ||
| + | <li> program: | ||
| + | <ul> | ||
| + | <li> 1. 98.0 °C 15 s | ||
| + | <li> 2. 98.0 °C 10 s | ||
| + | <li> 3. 72.0 °C 15 s | ||
| + | <li> 4. 72.0 °C 3 s | ||
| + | </ul> | ||
| + | <li> each step was repeated 30 times | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Gelelectrophoresis of the restriction digestion and PCR </b><br> | ||
| + | <ul> | ||
| + | <li> Gelelectrophoresis performed according to protocol 2 | ||
| + | <li> Changes to protocol | ||
| + | <ul> | ||
| + | <li> 70 ml 1 % agarose gel moulded | ||
| + | </ul> | ||
| + | |||
| + | <li> 1 µl staining solution added to 5 µl water control | ||
| + | <li> 1 µl staining solution added to 5 µl PCR-sample | ||
| + | <li> 2 µl staining solution added to 10 µl of digest | ||
| + | <li> Gel loaded according to following scheme | ||
| + | 1kb DNA-marker // digestion // water control // PCR | ||
| + | </ul> | ||
| + | |||
| + | <b> 4. Purification of the PCR fragment </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Analogous to Day 9/5. | ||
| + | <li> pellet was solved in 30 µl EB | ||
| + | </ul> | ||
| + | |||
| + | <b> 5. Restriction digestion of the YFP-PCR fragment </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> purified insert digested with XhoI Sal I | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="100%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>DNA</td><!-- Col 1 --> | ||
| + | <td>30 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>Sal I</td><!-- Col 1 --> | ||
| + | <td>2 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>XhoI</td><!-- Col 1 --> | ||
| + | <td>4 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td> Buffer O</td><!-- Col 1 --> | ||
| + | <td>10 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>ddH2O</td><!-- Col 1 --> | ||
| + | <td>54 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <ul> | ||
| + | <li> incubation (3 h, 37 °C)) | ||
| + | </ul> | ||
| + | |||
| + | <b> 6. Gelextraction of the digested vector </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> gelelectrophoresis (1.5 h) in 1 % agarose gel analogous to Day 9 4. | ||
| + | <li> band with scalpel removed and weighted = 390 mg | ||
| + | <li> three fold amount of QC added (1160 µl) | ||
| + | <li> analogue to Day 9 4. continued | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 15, 15/07/15 </h4> | ||
| + | |||
| + | <b>1. Purification of the digestion from day 14/5.</b><br> | ||
| + | <ul> | ||
| + | <li> Analogue to Day 9/5. | ||
| + | <li> Changes to Day 9/5. | ||
| + | <ul> | ||
| + | <li> 380 µl PB used | ||
| + | <li> eluted in 30 µl Elution Buffer | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Dephosphorylation of the vector </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> alkaline phosphatase mix created | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>TCPlac22 + insert XhoI Sal I digested</td><!-- Col 1 --> | ||
| + | <td>20 µl </td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>FAP buffer</td><!-- Col 1 --> | ||
| + | <td>3 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>FAP</td><!-- Col 1 --> | ||
| + | <td>1 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>H2O</td><!-- Col 1 --> | ||
| + | <td>6 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <ul> | ||
| + | <li> incubation (20 min, 37 °C) | ||
| + | <li> incubation (5 min, 75 °C) | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Ligation YCPlac22 + insert his3_rep_klein and YFP </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> 3 ligation samples prepared | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width=" 70%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>A)</td><!-- Col 1 --> | ||
| + | <td>4 µl vector, dephosphorylated (YCPlac22)</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>5 µl insert (YFP)</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>2 µl ligase buffer</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>1 µl T4 ligase</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>8 µl ddH2O</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <br> | ||
| + | <table border="0" width="53%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>B)</td><!-- Col 1 --> | ||
| + | <td>4 µl vector, dephosphorylated</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>2 µl ligase buffer</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>1 µl T4 ligase</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>12 µl ddH2O</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <br> | ||
| + | <table border="0" width="53%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>C)</td><!-- Col 1 --> | ||
| + | <td>4 µl vector, dephosphorylated</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>2 µl ligase buffer</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | |||
| + | <tr><!-- Row 4 --> | ||
| + | <td></td><!-- Col 1 --> | ||
| + | <td>14 µl ddH2O</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <ul> | ||
| + | <li> incubation (3 h, room temperature) | ||
| + | </ul> | ||
| + | |||
| + | <b> 4. Transformation of the ligation</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> performed analogue to Day 8 5. | ||
| + | <li> transformation plated as followed: | ||
| + | <ul> | ||
| + | <li> 200 µl, 100 µl, 50 µl of ligation on ampiciline plated | ||
| + | <li> 200 µl ligase + vector on ampiciline plated | ||
| + | <li> 200 µl vector on ampiciline plated | ||
| + | <li> 200 µl PBSC-Bluescript and H2O on ampiciline plated | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <h4>Day 16, 15/07/16</h4> | ||
| + | |||
| + | <b> 1. Picking of colonies </b><br> | ||
| + | <ul> | ||
| + | <li> 20 colonies of the ligation plate picked | ||
| + | <li> Each given in 5 ml 100 µg/ml ampiciline medium | ||
| + | <li> Incubation over night at 37 °C | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Retransformation of the ligation</b><br> | ||
| + | <ul> | ||
| + | <li> Repition of Day 15/3. (see above) | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 17, 15/07/17 </h4> | ||
| + | |||
| + | <b> 1. Miniprep of the over night culture </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Performed according to protocol 1 | ||
| + | </ul> | ||
| + | |||
| + | <b> 2. Restriction digestion from the Miniprep </b><br> | ||
| + | <ul> | ||
| + | <li> Restriction digest created | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="100%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <th align="left"> Miniprep</th> | ||
| + | <th align="left">1 µl </th><!-- Col 1 --> | ||
| + | <th align="left">Mastermix for 21 samples</th><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>Sal I</td> | ||
| + | <td>0.5 µl</td><!-- Col 1 --> | ||
| + | <td>10.5 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>XhoI</td> | ||
| + | <td>1 µl</td><!-- Col 1 --> | ||
| + | <td>21 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>Buffer 0</td> | ||
| + | <td>2 µl </td><!-- Col 1 --> | ||
| + | <td>42 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 5 --> | ||
| + | <td>ddH2O </td> | ||
| + | <td>15.5 µl</td><!-- Col 1 --> | ||
| + | <td>325.5 µl</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <br> | ||
| + | <ul> | ||
| + | <li> digestion (2 h, 37 °C) | ||
| + | </ul> | ||
| + | |||
| + | <b> 3. Gelelectrophoresis </b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> Gelelectrophoresis performed according to protocol 2 | ||
| + | <li> 4 µl staining solution was added to each digest | ||
| + | <li> 6 µl 1kb DNA-marker loaded | ||
| + | <li> scheme: | ||
| + | </ul> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td> 1kb DNA-marker // Miniprep 1 - 10 = Gel I </td> | ||
| + | <td> 1kb DNA-marker // Miniprep 11 - 20 = Gel II </td> | ||
| + | </tr> | ||
| + | <table> | ||
| + | |||
| + | <ul> | ||
| + | <li> Restriction sites XhoI and SalI are compatible, but after XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not. | ||
| + | <li> Restriction sites XhoI and SalI are compatible, but after XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not. | ||
| + | <li> Clones 1, 8, 9, 12, 14, 18 had an insert, which were similar to the expected size | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 18, 15/07/20</h4> | ||
| + | |||
| + | <b> 1. Overnight culture of S. cerevisiae K699 and 4196 </b><br> | ||
| + | <ul> | ||
| + | <li> Two colonies picked of each strain | ||
| + | <li> Inoculation of 5 ml YEPD-Medium each | ||
| + | <li> Incubation overnight at 28°C | ||
| + | </ul> | ||
| + | |||
| + | <h4>Day 19, 15/07/21</h4> | ||
| + | <b> 1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22-YFP</b><br> | ||
| + | |||
| + | <ul> | ||
| + | <li> over night culture dilitued 1:100 with MQ (10 µl suspension and 990 µl MQ) | ||
| + | <li> measurement 0D600: | ||
| + | </ul> | ||
| + | |||
| + | <table border="0" width="19%"> | ||
| + | <tr><!-- Row 1 --> | ||
| + | <td>699 A =</td><!-- Col 1 --> | ||
| + | <td>0.019</td> | ||
| + | <td>→ used</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 2 --> | ||
| + | <td>699 B =</td><!-- Col 1 --> | ||
| + | <td>0.014 </td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | <tr><!-- Row 3 --> | ||
| + | <td>4196 A =</td><!-- Col 1 --> | ||
| + | <td>0.108</td><!-- Col 2 --> | ||
| + | <td> → used</td> | ||
| + | </tr> | ||
| + | <tr><!-- Row 4 --> | ||
| + | <td>4196 B=</td><!-- Col 1 --> | ||
| + | <td>0.056</td><!-- Col 2 --> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ul> | ||
| + | <li> two flasks filled with YEPG next to Bunsenburner | ||
| + | </ul> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td>A: 25 ml</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>B: 25 ml</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ul> | ||
| + | |||
| + | <li> 4,46 ml added to flask A suspension 699 oD600 = 0,208 | ||
| + | <li> 0.58ml added to flask B suspension 4196 oD600 = 0,193 | ||
| + | <li> 3 h at 30 °C and incubated while shaking | ||
| + | <li> 25 ml yeast culture from the flask given in 50 ml falcon | ||
| + | <li> oD600 determined | ||
| + | </ul> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td>699 = 0.543</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>4196 = 0.503</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | <ul> | ||
| + | <li> Centrifugation (5 min, 3500 rpm, room temperature) | ||
| + | <li> Supernatant discarded | ||
| + | <li> Contamination in 4196 suspension detected => Sample discarded | ||
| + | <li> Pellet resuspended in 25 ml ddH2O | ||
| + | <li> Pelletised at 3500 rpm and room temperature | ||
| + | <li> Supernatant discarded | ||
| + | <li> Pellet in 1 ml of 100 mM LiAc resuspended and given in 1.5 reaction tubes | ||
| + | <li> Centrifugation (10 s, 3500 rpm, room temperature) | ||
| + | <li> Supernatant discarded | ||
| + | <li> Pellet resuspended in 500 µl 100 mM LiAc | ||
| + | <li> For each transformation 50 µl cell suspension (6) transferred in 1.5ml ml reaction tubes | ||
| + | <li> Centrifugated for 10 s and supernatant discarded | ||
| + | <li> Mix for transformation added (adhered to order as written below) | ||
| + | <ul> | ||
| + | <li>> 240 µl 50 % PEG 3350 | ||
| + | <li>> 36 µl 1 M LiAc | ||
| + | <li>> 5 µl carrier DNA (10mg/ml) | ||
| + | <li>> 5µl ddH2O (negative control)/ YCplac22-YFP 1, 8, 9, 12, 14 and 18 | ||
| + | <li>> 64 µl of ddH2O | ||
| + | </ul> | ||
| + | <li> Sample vortexed until pellet was resuspended | ||
| + | <li> Incubation at room temperature (30 °C) | ||
| + | <li> Heat shock for 20 min at 42 °C | ||
| + | <li> Centrifugated for 10 s, | ||
| + | <li> Pellet resuspended in 400 µl H2O | ||
| + | <li> 200 µl and 100µl of transformation plated on mediaplates without tryptophan | ||
| + | <li> Incubated (72 h, 30 °C) | ||
| + | </ul> | ||
| + | |||
| + | <h4> Day 20, 15/07/24 </h4> | ||
| + | <b>1. Convokal Microskopy</b><br> | ||
| + | <ul> | ||
| + | <li> One colony per construct picked and diluted in Water | ||
| + | <li> Microscopy → see result section | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| + | <p>1 von Filip</p> | ||
| + | <p>1 von Vlady (schon auf Studon)</p> | ||
| + | <p>1 von Frederike</p> | ||
</html> | </html> | ||
{{FAU_Erlangen_footer}} | {{FAU_Erlangen_footer}} | ||
Revision as of 16:40, 18 September 2015
Yeast transformation with YFP
Yeast transformation with YFP
Day 1, 15/06/25:
1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of hostlab's strain collection- Strains taken from -80°C freezer were spread out on YPED-Medium plates
- Incubation for 3 days at 26°C
- 1 µl DNA Stocksolution was given onto 50µl bacteria suspension
- Incubation on ice for 30 minutes
- Heatshock at 42°C for 90 seconds
- Incubation on ice for 2 minutes
- Addition of 500µl SOC-Medium
- Incubation at 37°C for 60 minutes
- 100µl of suspension wase plated on agarplates containing ampicilin
- Incubation at 37° C over night
Day 2, 15/06/26:
1. Picking colonies for overnight cultures (ONK)- Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000)
- Afterwards incubation overnight at 37°C
Day 3, 15/06/29:
1. DNA-preparation via alkaline Lysis- Experimental procedure according to " Protocol 1: Alkaline Lysis "
- Changes to protocol:
- No centrifugation of ONK
- Two 2ml reaction tubes filled with ONK instead
- Two yeast colonies picked out of YPED
- Medium plates from Day 1 (see day 1/1.)
- Clone 1 named A
- Clone 2 named B
Day 4, 15/06/30:
1. Restriction digest of the obtained DNA-Solutions- Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table
| Reagent | Volume for one sample | Mastermix for four samples |
|---|---|---|
| EcoRI | 0.5 µl | 2 µl |
| EcoRV | 0.5 µl | 2 µl |
| Tango Buffer | 4 µl | 16 µl |
| Add 20µl ddH2O | 14 µl | 56 µl |
| Σ | 19 µl | 76 µl |
- 19 µl out of the mastermix were transferred to three 1.5 ml reaction tubes
- 1 µl YCplac22/YEplac181/YIplac204 solution obtained in day 3/1 were given in one mastermix tube each
- 1µl K80100 solution obtained in 3/1 was added to following reagents:
| Reagent | Volume for one sample |
|---|---|
| PstI | 0.5 µl |
| Buffer O | 2 µl |
| Add 20µl ddH2O | 16.5 µl |
| Σ | 19 µl |
- Digests were incubated at 37°C for 3 h
- Gelectrophoresis was executed according to "protocol 2: Gelelectrophoresis "
- 2 µl 6x staining solution were given unto 10 µl digest
- Samples were loaded onto the gel according to following scheme
⇒ 1kb-DNA-Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\1kb DNA-marker (6µl) - Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes
Day 5, 15/07/01:
1. Repetition of restriction digest for Ycplac 204 and K801000 digested- Mastermix created for BBa_K801000 and YIplac204
Reagent Volume for one sample Mastermix for three samples Eco RI 0.5 µl 1.5 µl EcoRV 0.5 µl 1.5 µl Tango Buffer 4 µl 12 µl Add 20µl ddH2O 13 µl 39 µl Σ 18 µl 54 µl
- 2µl of obtained DNA
- Solution were transfered to 18 µl of mastermix
- Incubation at 37°C for 3 hours
- Gelelectrophoresis was conducted according to protocol 2
- 2 µl of 6x staining solution were added onto 10 µl of digest
- Samples were loaded in pockets according to following scheme
⇒ 6µl 1kb-DNA-marker// YIplac204 digested (15/07/01) // K801000 digested(15/07/01)//YCplac22 (15/06/30)// K801000(15/06/30)// YIplac204 (15/06/30)//empty//YEplac181 (15/06/30)// empty // 6µl 1kb-DNA-marker
- Added following substances in two 1 liter flasks:
- 3.35g Difco yeast nitrogen base 2/o aminoacid
- 5.5 g CAA vitamin assay
- 10 g Glucose
- 83.0 mg Tyrosin
- Uracil
- Adenin
- mix
- 50.5 mg Leucin
- 22g Agar
- Added following substances in two 1 liter flasks
- 5.3g yeast extract
- 11g Bacopepton
- 10g Glucose
- 22.7 mg Adenin
- 11g Agar
- Two times 5 ml ampicilin medium (80µg/ml) inoculated with 2 ampicilline ressistant bacteria colonies picked of plate from day 1/2.
- Incubation at 37°C over night
Day 6, 15/07/02:
1. Moulding of Agar-plates- Added 500ml destilled Water to each Flask of day 5/2. and day 5/3.
- Short mixing with stirring bar
- Flask autoclaved
- Stirring with stirring bar for 20 minutes at room temperature
- Moulding of plates underneath a laminar flow hood
- Conducted analogous to day 3/1. according to protocol 1
- Mastermix created to digest 2 µl DNA solution
Reagent Volume for one sample Mastermix for three samples EcoRV 0.5 µl 1.5µl Buffer R 2.0 µl 6 µl Add 20µl H2O 15.5µl 46.5µl Σ 18µl 18µl
- 2 µl DNA-preparation of each dublicat of YIp204 were added onto 18µl mastermix
- Incubated for 3 hours at 37°C
- Electrophoresis was conducted according to protocol 2
- Added 4µl 6x staining buffer to each digest after incubation time
- Added 4µl 6x staining buffer to 20µl undigest DNA
- Preperation
- Loaded gel according to following scheme
⇒ 6µl 1kb-DNA-marker\\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested - DNA-fragments of unknown origin were found
- Repitition of experiment needed
- 3 ml YEPD Medium was given to each of 4 reaction tubes
- Two yeast colonies of 4196 and K669 were picked
- Incubation at 30°C over night
- Mix to digest 10 µl YIplac204 DNA obtained in preparation 3.1 for transformation created
Reagent Volume for one sample Eco RV 1 µl Buffer R 5 µl Add 20µl ddH2O 34 µl Σ 40 µl
- Incubation over night at 37°C
- 5 ml of ampiciline-medium (80µg/ml) were inoculated with one colony obtained of experiment day 1/2.
- Incubation at 37°C overnight
Day 7, 15/07/03
1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22 and Ylplac204- over night culture diluted 1:100 with MQ (10 µl suspension and 990 µl MQ)
- measurement 0D600:
- 699 A = 0.203
- 699 B = 0.143 → used
- 7196 A = 0.12
- 4196 B = 0.139 → used
- two flasks filled with YEPG next to Bunsenburner
- A: 50 ml
- B: 25 ml → Media (wrong estimate)
- in flask A 2 ml suspension 699 oD600 = 0.30
- in flask B 1 ml suspension 4196 oD600 = 0.293
- 3 h at 30 °C and incubated while shaking
- Experiment conducted according to protocol 1
- Experiment conducted according to protocol 2
- Changes to protocol: → 1.2 % agarose gel
- 5 µl of overnight digest added to 1 µl 6x staining buffer
- loaded onto gel according to following scheme ⇒ 6 µl 1kb-DNA-marker \\ overnight digest
- After the photo was taken DNA-Preparation from day 7/2. loaded on same gel
- Scheme : 6 µl 1kb-DNA-marker //overnight digest //empty// marker // Miniprep
- 3.5 µl 3M NaAC added to DNA solution
- 1.5 µl Glycogen (Thermo Scientific) added
- 100µl 95% Ethanol added
- Incubation for 5 minutes at room temperature
- Centrifugation (fullspeed, 5 min, room temperature)
- DNA appears as a small pellet
- Supernatant removed
- Pellet washed in two volumes (240 µl) EtOH 70 %
- Dried at room temperature for 30 min
- DNA solved in 5 µl TE
- 25 ml yeast culture from the flask given in 50 ml falcon
- oD determined
- 699 = 0.76
- 4196 = 0.75
- Centrifugation (5 min, 3500 rpm, room temperature)
- Supernatant discarded
- Pellet resuspended in 25 ml ddH2O
- Pelletised at 3500 rpm and room temperature
- Supernatant discarded
- Pellet in 1 ml of 100 mM LiAc resuspended and given in reaction tubes
- Centrifugation (10 s, 3500 rpm, room temperature)
- Supernatant discarded
- Pellet resuspended in 500 µl 100 mM LiAc
- For each transformation 50 µl cell suspension (4) transferred in 1.5ml ml reaction tubes
- Centrifugated for 10 s and supernatant discarded
- All samples of 4196 discarded (too few DNA)
- Mix for transformation added (adhered to order as written below)
- 240 µl 50 % PEG 3350
- 36 µl 1 M LiAc
- 5 µl carrier DNA (10mg/ml)
- 4 µl YEP122 (positive control) / 5 µl YIP204/ 3µl YCplac22/ 5µl ddH2O (negative control) → 65 µl of ddH2O to YEplac122 → 360 µl
→ 64 µl of ddH2O to YIplac204 → 360 µl
→ 66 µl ddH2O to YCplac22 → 360 µl
→ 64 µl ddH2O to negative control → 360 µl - Sample vortexed until pellet was resuspended
- Incubation at room temperature (30 °C)
- heat shock for 20 min at 42 °C
- centrifugated for 10 s, pellet resuspended in 400 µl H2O
- YEplac122 200 µl plated
- YIplac204 400 µl plated
- YCplac22 200 µl plated
- negative control 200 µl plated
- incubated (72 h, 30 °C)
1. Examination of 7/4.
- Colonies grown!
- Transformation works
2. Resuspension of IDT geneBricks his_spacer adh1 and his_rep_klein
- 500 ng in 50 µl TE given (c = 10 ng/ml)
- incubated (50 °C, 20 min)
- vortexed and centrifugated (10 s at fullspeed)
3. Restriction digest for ligation of his_spacer adh1 and his_rep_klein
- calculations for needed amount of DNA via following formula:
m(plasmid) * lengt (insert)/length(Vector)*5 → factor 5 is based on experience
Thus following values were calculated:
Insert his_rep_klein for cloning in YIplac204 =200 ng (plasmid) * 700 bp/3500 bp *5 = 200ng Insert his_rep_klein for cloning in YCplac22 =200 ng (plasmid) * 700 bp/4850 bp *5 = 144 ng Insert his_spacer_adh1 for cloning in YEplac181 =200 ng (plasmid) * 500 bp/5740 bp *5 = 86ng Insert his_spacer_adh1 for cloning in K801000 =200 ng (plasmid) * 500 bp/5500 bp *5 = 90ng
- following restriction digests were constructed:
- insert his_spacer_adh1/ insert His_Rep_klein
DNA 40µl MM for 3 samples EcoRI 2µl 6µl PstI 2µl 6µl Buffer 0 20µl 60µl add 200µl ddH2O 136 µl 408 µl
- Vector YEplac181/ YCplac22/ YEplac204 (7/3)
DNA 5 µl MM for 4 samples RNAse 1 µl 4 µl EcoRI 1 µl 4 µl PstI 1 µl 4 µl Buffer O 5 µl 20 µl add 50µl ddH2O 37 µl 148 µl
- Vector K801000
DNA 40 µl RNAse 1 µl EcoRI 2 µl PstI 2 µl Buffer O 10 µl add 100µl ddH2O 45 µl
- large volumes were chosen because of the high EDTA-concentration
- over night incubation at 37 °C 4. CloneJet (Thermo Scientific) PCR-cloning with his3_rep_klein and his_spacer_adh1
- as a backup the following plasmids were generated using the CloneJET PCR Cloning Kit
2 µl 10 x ligase buffer 2.5 µl DNA solution 1 µl pjet-vector add 19 µl ddH2O 13.5µl 1 µl T4Ligase - Incubation (ca. 10 min)
- DH5alpha E. coli unfreezed
- Each 5 µl from Day8 4. given on top of 50 µl competent cells
- As a positive control 1µl PBSC
- Bluescript was given on top of 50 µl E.coli
- As a negative control no changes were applied to 50 µl E. coli
- incubate for about 15 min on ice
- heat shock for 90 s
- 2 min on ice
- 500 µl SOC medium added
- incubated (45 min, 37 °C)
- centrifugation (2 min, 7000 rpm)
- remove 450 µl supernatant
- E.coli in remained 100 µl resuspended
- 100 µl plated on ampiciline plates
Day 9, 15/07/07
1. Analysis of over night digest from Day 8/3.
- Electrophoresis was conducted according to protocol 2
- The gel was loaded with 10 % digest volume
- 6x staining buffer was added according to volume (given in Brackets)
YEplac181/ YCplac22/YIplac204
5 µl
(1 µl)
Bba_K801000
10 µl
(2 µl)
Inserts (his3_rep_klein/ his_spacer_adh1
20 µl
(4 µl)
- Pockets were loaded according to following scheme
⇒ 1 kb DNA-marker //YEplac181//YCplac22//YIplac204//K801000//his_spacer_adh1//his3_rep_klein
- 40 µl plasmid solution 204 obtained out of Day 7 2. digested
DNA 40 µl EcoRI 2 µl PstI 2 µl Buffer O 20 µl H2O 136 µl Σ 200 µl - incubation (2h, 37 °C)
- Gelelectrophoresis conducted according to protocol 2
- Middlepocket of gel loaded with 45 µl YCplac22 Day 8 3.according to following scheme:
⇒ 6µl 1 kb DNA - marker // empty // YCplac22 // empty
- Changes to protocol:
- large comb used
- run for 2h at 50V
- Gel fragment weighted: 470 mg
- 3 times the volume QC-buffer added → 1410 µl QC-buffer added
- 10 min at 50 °C gel dissolved
- After 3 min and 7 min for 5-7 s vortexed
- 450 µl isopropanol added
- Sample inverted and shortly vortexed
- 800 µl given on speedcolumn with wastetube
- Centrifugation (13300 rpm, 1 min) → flowthrough discarded
- Step 3 times repeated
- 0,75 ml PE buffer added on column for cleaning
- Centrifugation (13300 rpm, 1 min)
- Flowthrough discarded
- Centrifugation (13300 rpm, 1 min)
- Flowthrough discarded
- Column transferred on 1.5 ml tube
- 30 µl Elutionbuffer added on column
- Centrifugation (13300 rpm, 1 min)
- Eluate used for further experiments
- 180 µl sample given to 900 µl PB buffer given
- 800 µl Sample given on column
- column centrifugated (13300 rpm, 1 min)
- remaining 280 µl given on column
- column centrifugated at 13300 rpm
- both times flowthrough was discarded
- column loaded with 750 µl PE
- centrifugation (13300 rpm, 1 min)
- flowthrough discarded
- centrifugation (13300 rpm, 1 min)
- flowthrough discarded
- column transferred on 1.5 ml Eppi
- 30 µl EB (elution buffer) given
- centrifugation (13300 rpm, 1 min)
- eluate used for further experiments
- Gelelectrophoresis according to protocol 2: Electrophoresis
- Gel loaded with samples according to following scheme:
- Changes to protocol:
- Gelelectrophoresis for 45 min
- note: insert was added after 20 min
- 2 colonies on plate 204, Pjet_His_spacer, Pjet_reporter_klein picked
- Each were given into 5 ml 80 µg/µl ampiciline medium given
- over night incubation at 37 °C
Day 10, 15/07/08:
1. Ligation of the linearised vector YCplac22 and the his3_rep_klein
- 3 samples for ligation generated
- ligation with insert
- 3 µl vector (YCPlac22) linearized
- 14 µl insert (His3_rep_klein/ his_spacer_adh1) linearized
- 2 µl ligase buffer
- 1 µl T4 Ligase
- Religation control without insert
- 3 µl vector (YCPlac22) linearized
- 14 µl ddH2O
- 2 µl ligase buffer
- 1 µl T4 Ligase
- control for complete digestion
- 3 µl vector (YCPlac22) linearized
- 15 µl H2O
- 2 µl ligase buffer
- ligation incubated for 3 h at room temperature
- ligation with insert
- 2 ml over night culture transferred to 2 ml
- reaction tubes
- centrifugation (7000 rpm, 5 min)
- supernatant discarded
- 2 ml from over night culture transferred in the same reaction tubes as their precursors to increase the amount of bacteria
- centrifugation (7000 rpm, 5 min)
- supernatant discarded
- DNA
- Preparation conducted as given by Quia
- Gen Miniprep instruction
- Elution in 50 µl elution buffer
- Digest prepared
1 µl DNA from the miniprep(see above) MM for 7 samples à 19 µl EcoRI 0.5 µl 3.5 µl PstI 0.5 µl 3.5 µl Buffer O 2 µl 14 µl ddH2O 16 µl 112 µl - digestion for 3 h at 37 °C
- TY-Medium with agar heated
- Cooling while mixing
- Addition of 50 µl Chloramphenicol (100 mg/ml)
- c_end= 10µg/ml
- Moulding of plates
- 40µl 2% x-gal spread on 6 ampicilin enriched plates
- 40µl IPTG spread on the dried plates
- 40µl dH2O spread on the dried plates
- Each of folowing DNA
- samples added to 50 µl DH5α on ice
- 5µl ligation from 10.1
- 5µl ligase + vectorligation from 10.1
- 5µl vector + ligase buffer solution from 10.1
- 1µl pBluescript
- 5µl H2O
- 2.5µl EYFP (BBa_E2030)
- Transformation analogous to 8.5
- Transformed E. coli samples spread on ampiciline plates as followed
- 100 µl ligation
- 200 µl ligation
- 200 µl ligase + vector
- 200 µl pBluescript
- 200 µl H2O
- Remaining transformed bacteria spread on chloramphenicole agar plates
- 200 µl EYFP (BBa_E2030)
- 200 µl H2O
- Gelelectrophoresis conducted according to protocol 2
- 20 µl taken from earlier digest (see above) and added to 4µl Staining Solution
- Gel loaded according to following scheme:
Day 11, 15/07/09
1. Cultures picked for over night culture
- 12 colonies taken from 100 µl plate (compare Day 10 6.)
- inoculation of 2 ml 80 µg/ml ampiciline medium
- 2 colonies picked from YFP
- plate
- 5 ml 25 µg/ml Canamycin medium inoculated
- over night incubation at 37 °C
Day 12, 15/07/10
1. Miniprep of the over night culture from day 11/1. (see above)
- conducted according to protocol 1
- eluted in 30µl 1x TE
- Restriction for all DNA-preparations (14 samples)
DNA Solution 3 µl MM for 15 PstI 0.5 µl 7.5 µl EcoRI 0.5 µl 7.5 µl Buffer O 2 µl 30 µl ddH2O 14 µl 210 µl - incubation (1.5 h, 37 °C)
- Gelelectrophoresis performed according to protocol 2
- Changes to protocol:
- Volume for two 1% agarosegels produced 170 ml (100 ml+70 ml)
- 13,6 µl Roth Ethidiumbromide added
- Samples from Day 12 2. (see above) were added to 4 µl staining solution
- Gels were loaded as followed:
Gel I: 1kb DNA-marker// YCPlac22 + insert 1 // 2 // 3 // 4 // 5 // 6 // 7 // 8 //
Gel II: 1kb DNA-marker // YCPlac22 + insert 9 // 10 // 11 // 12 // empty// YFP clone 1 // YFP clone 2// stamdard// - Results not expected ratio insert vector seems tilted
- Over night culture of all samples analogous to Day 11/1.
Day 13, 15/07/11
1. Miniprep via Quiagen column
- 5 ml over night culture from day 12/3. centrifuged (7000 rpm, 5min) in 2 ml Eppis
- Quiagen Miniprep analogous to 10/2. performed
- DNA-Solutions obtained in Day 13/1. (see above) EcoRI PstI digested
- Mastermix created
single sample Mastermix for 13 Samples 1 µl DNA - 0.5 µl EcoRI 6.5 µl EcoRI 0.5 µl PstI 6.5 µl PstI 2 µl Buffer0 26 µl Buffer0 16 µl ddH2O 208 µl ddH2O - incubation (2 h, 37 °C)
- Gelelectrophoresis conducted according to protocol 2
- 4 µl staining solution added to each of the 12 samples from experiment Day 13/2. (see above)
- 24 µl loaded on gel according to following scheme
- 1kb DNA-marker //
- YCPlac 22 + insert 1 //
- YCPlac 22 + insert 2 //
- YCPlac 22 + insert 3 //
- YCPlac 22 + insert 4 //
- ...
- YCPlac 22 + insert 12 //
Day 14, 15/07/14
1. Restriction digestion XhoI, Sal I from YCPlac22 + insert10 (day 13/13)
- restriction digestion conducted
DNA YCPlac22 + insert10 from day 13 20 µl Sal I 2 µl XhoI 4 µl Buffer 0 10 µl ddH2O 64 µl - incubation (2 h, 37 °C)
Sample water control H2O 71 µl 73 µl 5 x buffer 20 µl 20 µl template 2 µl - Forward Primer 2 µl 2 µl Reverse Primer 2 µl 2 µl Fusion Polymerase 1 µl 1 µl Σ 100 µl 100 µl - program:
- 1. 98.0 °C 15 s
- 2. 98.0 °C 10 s
- 3. 72.0 °C 15 s
- 4. 72.0 °C 3 s
- each step was repeated 30 times
- Gelelectrophoresis performed according to protocol 2
- Changes to protocol
- 70 ml 1 % agarose gel moulded
- 1 µl staining solution added to 5 µl water control
- 1 µl staining solution added to 5 µl PCR-sample
- 2 µl staining solution added to 10 µl of digest
- Gel loaded according to following scheme 1kb DNA-marker // digestion // water control // PCR
- Analogous to Day 9/5.
- pellet was solved in 30 µl EB
- purified insert digested with XhoI Sal I
DNA 30 µl Sal I 2 µl XhoI 4 µl Buffer O 10 µl ddH2O 54 µl - incubation (3 h, 37 °C))
- gelelectrophoresis (1.5 h) in 1 % agarose gel analogous to Day 9 4.
- band with scalpel removed and weighted = 390 mg
- three fold amount of QC added (1160 µl)
- analogue to Day 9 4. continued
Day 15, 15/07/15
1. Purification of the digestion from day 14/5.
- Analogue to Day 9/5.
- Changes to Day 9/5.
- 380 µl PB used
- eluted in 30 µl Elution Buffer
- alkaline phosphatase mix created
TCPlac22 + insert XhoI Sal I digested 20 µl FAP buffer 3 µl FAP 1 µl H2O 6 µl - incubation (20 min, 37 °C)
- incubation (5 min, 75 °C)
- 3 ligation samples prepared
A) 4 µl vector, dephosphorylated (YCPlac22) 5 µl insert (YFP) 2 µl ligase buffer 1 µl T4 ligase 8 µl ddH2O
B) 4 µl vector, dephosphorylated 2 µl ligase buffer 1 µl T4 ligase 12 µl ddH2O
C) 4 µl vector, dephosphorylated 2 µl ligase buffer 14 µl ddH2O - incubation (3 h, room temperature)
- performed analogue to Day 8 5.
- transformation plated as followed:
- 200 µl, 100 µl, 50 µl of ligation on ampiciline plated
- 200 µl ligase + vector on ampiciline plated
- 200 µl vector on ampiciline plated
- 200 µl PBSC-Bluescript and H2O on ampiciline plated
Day 16, 15/07/16
1. Picking of colonies
- 20 colonies of the ligation plate picked
- Each given in 5 ml 100 µg/ml ampiciline medium
- Incubation over night at 37 °C
- Repition of Day 15/3. (see above)
Day 17, 15/07/17
1. Miniprep of the over night culture
- Performed according to protocol 1
- Restriction digest created
Miniprep 1 µl Mastermix for 21 samples Sal I 0.5 µl 10.5 µl XhoI 1 µl 21 µl Buffer 0 2 µl 42 µl ddH2O 15.5 µl 325.5 µl
- digestion (2 h, 37 °C)
- Gelelectrophoresis performed according to protocol 2
- 4 µl staining solution was added to each digest
- 6 µl 1kb DNA-marker loaded
- scheme:
1kb DNA-marker // Miniprep 1 - 10 = Gel I 1kb DNA-marker // Miniprep 11 - 20 = Gel II - Restriction sites XhoI and SalI are compatible, but after XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not.
- Restriction sites XhoI and SalI are compatible, but after XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not.
- Clones 1, 8, 9, 12, 14, 18 had an insert, which were similar to the expected size
Day 18, 15/07/20
1. Overnight culture of S. cerevisiae K699 and 4196
- Two colonies picked of each strain
- Inoculation of 5 ml YEPD-Medium each
- Incubation overnight at 28°C
Day 19, 15/07/21
1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22-YFP
- over night culture dilitued 1:100 with MQ (10 µl suspension and 990 µl MQ)
- measurement 0D600:
699 A = 0.019 → used 699 B = 0.014 4196 A = 0.108 → used 4196 B= 0.056 - two flasks filled with YEPG next to Bunsenburner
A: 25 ml B: 25 ml - 4,46 ml added to flask A suspension 699 oD600 = 0,208
- 0.58ml added to flask B suspension 4196 oD600 = 0,193
- 3 h at 30 °C and incubated while shaking
- 25 ml yeast culture from the flask given in 50 ml falcon
- oD600 determined
699 = 0.543 4196 = 0.503 - Centrifugation (5 min, 3500 rpm, room temperature)
- Supernatant discarded
- Contamination in 4196 suspension detected => Sample discarded
- Pellet resuspended in 25 ml ddH2O
- Pelletised at 3500 rpm and room temperature
- Supernatant discarded
- Pellet in 1 ml of 100 mM LiAc resuspended and given in 1.5 reaction tubes
- Centrifugation (10 s, 3500 rpm, room temperature)
- Supernatant discarded
- Pellet resuspended in 500 µl 100 mM LiAc
- For each transformation 50 µl cell suspension (6) transferred in 1.5ml ml reaction tubes
- Centrifugated for 10 s and supernatant discarded
- Mix for transformation added (adhered to order as written below)
- > 240 µl 50 % PEG 3350
- > 36 µl 1 M LiAc
- > 5 µl carrier DNA (10mg/ml)
- > 5µl ddH2O (negative control)/ YCplac22-YFP 1, 8, 9, 12, 14 and 18
- > 64 µl of ddH2O
- Sample vortexed until pellet was resuspended
- Incubation at room temperature (30 °C)
- Heat shock for 20 min at 42 °C
- Centrifugated for 10 s,
- Pellet resuspended in 400 µl H2O
- 200 µl and 100µl of transformation plated on mediaplates without tryptophan
- Incubated (72 h, 30 °C)
Day 20, 15/07/24
1. Convokal Microskopy
- One colony per construct picked and diluted in Water
- Microscopy → see result section
1 von Filip
1 von Vlady (schon auf Studon)
1 von Frederike
- following restriction digests were constructed: