Difference between revisions of "Team:FAU Erlangen/Tour32"
Adrian FAU (Talk | contribs) |
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<tr> | <tr> | ||
− | <td> Add 20µl ddH2O</td> | + | <td> Add 20µl ddH2O </td> |
<td> 13 µl </td> | <td> 13 µl </td> | ||
<td> 39 µl </td> | <td> 39 µl </td> | ||
Line 222: | Line 222: | ||
<tr> | <tr> | ||
− | < | + | <th> Σ </th> |
<td> 18 µl </td> | <td> 18 µl </td> | ||
<td> 54 µl </td> | <td> 54 µl </td> | ||
Line 275: | Line 275: | ||
− | Day 6, 15/07/02: | + | <h2> Day 6, 15/07/02: </h2> |
− | + | <h3> 1. Moulding of Agar-plates </h3> | |
− | <li> Added 500ml destilled Water to each Flask of 5 | + | <ul> |
+ | <li> Added 500ml destilled Water to each Flask of day 5/2. and day 5/3. | ||
<li> Short mixing with stirring bar | <li> Short mixing with stirring bar | ||
<li> Flask autoclaved | <li> Flask autoclaved | ||
<li> Stirring with stirring bar for 20 minutes at room temperature | <li> Stirring with stirring bar for 20 minutes at room temperature | ||
<li> Moulding of plates underneath a laminar flow hood | <li> Moulding of plates underneath a laminar flow hood | ||
+ | </ul> | ||
+ | <h3> 2. DNA<li>Preperation of overnight culture (see day 5/4.)</h3> | ||
+ | <ul> | ||
+ | <li> Conducted analogous to day 3/1. according to protocol 1 | ||
+ | </ul> | ||
+ | <h3> 3. Digest of obtained DNA </h3> | ||
+ | <ul> | ||
+ | <li> Mastermix created to digest 2 µl DNA solution | ||
− | + | <table> | |
− | < | + | <tr> |
+ | <th> Reagent </th> | ||
+ | <th> Volume for one sample</th> | ||
+ | <th> Mastermix for three samples</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> EcoRV </td> | ||
+ | <td> 0.5 µl</td> | ||
+ | <td> 1.5µl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> Buffer R</td> | ||
+ | <td> 2.0 µl</td> | ||
+ | <td> 6 µl </td> | ||
+ | </tr> | ||
− | + | <tr> | |
+ | <td> Add 20µl H2O </td> | ||
+ | <td> 15.5µl </td> | ||
+ | <td> 46.5µl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> Σ </th> | ||
+ | <td> 18µl </td> | ||
+ | <td> 18µl </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<li> 2 µl DNA DNA<li>preperation of each dublicat of YIp204 were added onto 18µl mastermix | <li> 2 µl DNA DNA<li>preperation of each dublicat of YIp204 were added onto 18µl mastermix | ||
Line 308: | Line 336: | ||
<li> Added 4µl 6x staining buffer to 20µl undigest DNA<li> Preperation | <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 | ||
− | + | ||
− | <li> DNA | + | ⇒ 6µl 1kb-DNA-marker\\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested\\ |
+ | |||
+ | <li> DNA-fragments of unknown origin were found | ||
Bilder unter 150702a | Bilder unter 150702a | ||
http://www.studon.uni<li>erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= | http://www.studon.uni<li>erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= | ||
+ | </ul> | ||
− | + | <h3> 4. Overnight culture of strains 4196 and K699 for transformation </h3> | |
+ | <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 30°C over night | <li> Incubation at 30°C over night | ||
+ | </ul> | ||
− | + | <h3> 5. Restriction digest of Vector DNA for the transformation </h3> | |
− | + | ||
<li> Mix to digest 10 µl YIplac204 DNA obtained in preparation 3.1 for transformation created | <li> Mix to digest 10 µl YIplac204 DNA obtained in preparation 3.1 for transformation created | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | < | + | <table> |
+ | <tr> | ||
+ | <th> Reagent </th> | ||
+ | <th> Volume for one sample </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> Eco RV </td> | ||
+ | <td>1 µl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> Buffer R </td> | ||
+ | <td> 5 µl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> Add 20µl ddH2O </td> | ||
+ | <td> 34 µl </td> | ||
+ | </tr> | ||
− | + | <tr> | |
+ | <th> Σ </th> | ||
+ | <th> 40 µl </th> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <li> Incubation over night at 37°C | ||
+ | </ul> | ||
+ | |||
+ | <h3> 6. Overnight culture of YIplac204 </h3> | ||
+ | |||
+ | <ul> | ||
<li> 5 ml of ampiciline<li>media (80µg/ml) were inoculated with one colony obtained of experiment Day1 2. (p.: 2) | <li> 5 ml of ampiciline<li>media (80µg/ml) were inoculated with one colony obtained of experiment Day1 2. (p.: 2) | ||
<li> Incubation at 37°C overnight | <li> Incubation at 37°C overnight | ||
+ | </ul> | ||
− | + | <h2> Day 7, 15/07/03 </h2> | |
− | Day 7, 15/07/03 | + | |
Revision as of 10:30, 17 September 2015
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
2. Transformation of Vectors YCplac22, YEplac 181 and YIplac204 in DH5&alpha E. coli
- 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
2. Picking of yeast colonies
- 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
2. Gelelectrophoresis of digested DNA
- 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\\DNA - 1kb
- Marker (6µl)
- Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes <\ul> hier Bilder 150630a
- c einfügen (http://www.studon.uni
- erlangen.de/studon/ilias.php?ref_id=1325854&cmd=frameset&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI) mit folgenden Bildunterschriften Figure 1: Gelphoto taken 1 hour after start.. Estimated fragment sizes were for YCpla22 (total: 4854) = 2171bp and 2683bp, YEplac181(total: 5741 bp)= 3170bp and 2571bp, YIplac204 (total:3545)= 2381 bp and 880 bp as well as Bba_K801000 (total: 4923 bp)= 3043bp, 1390 and 490bp. All except YIplac204 and Bba_K801000 show estimated dna bands. Picture was digitally altered by removing stains in the left corner and including RNA
- Description as well as cropping.
Figure 2: Gelphoto after 1h 40 minutes.
Figure 3: Gelphoto after 2h 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<\th> 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 Bild 150701a aus http://www.studon.uni
- erlangen.de/studon/goto.php?target=fold_1325854 Figure 4: Photo of gelelectrophoresis at 120 V after 50 minutes.
2. Preparation of S. Cerevisiae tryptophan negative plates
- 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
3. Preparation of S. Cerevisiae full media plates
- Added following substances in two 1 liter flasks → 5.3g yeast extract → 11g Bacopepton → 10g Glucose → 22.7 mg Adenin → 11g Agar
4. Overnightculture of YIplac204 positive bacteria
- 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
2. DNA
- Mastermix created for BBa_K801000 and YIplac204
- Preperation of overnight culture (see day 5/4.)
- Conducted analogous to day 3/1. according to protocol 1
3. Digest of obtained DNA
- 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 DNA
- preperation 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 Bilder unter 150702a http://www.studon.uni
- erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode=
4. Overnight culture of strains 4196 and K699 for transformation
- 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
5. Restriction digest of Vector DNA for the transformation
- 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
6. Overnight culture of YIplac204
- 5 ml of ampiciline
- media (80µg/ml) were inoculated with one colony obtained of experiment Day1 2. (p.: 2)
- Incubation at 37°C overnight
Day 7, 15/07/03
\subtitle 1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22 and Ylplac2041 von Adrian
1 von Filip
1 von Vlady (schon auf Studon)
1 von Frederike