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+ | |||
+ | <h1> Yeast transformation with YFP </h1> | ||
+ | |||
+ | <h2>Day 1, 15/06/25:</h2> | ||
+ | |||
+ | <h3> | ||
+ | 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of hostlab's strain collection | ||
+ | </h3> | ||
+ | |||
+ | |||
+ | <li> Strains taken from -80°C freezer were spread out on YPED-Medium plates | ||
+ | <li> Incubation for 3 days at 26°C | ||
+ | |||
+ | |||
+ | <h3> | ||
+ | 2. Transformation of Vectors YCplac22, YEplac 181 and YIplac204 in DH5&alpha E. coli | ||
+ | </h3> | ||
+ | |||
+ | <ul> | ||
+ | <li> 1 µl DNA Stocksolution was given onto 50µl bacteria suspension | ||
+ | <li> Incubation on ice for 30 minutes | ||
+ | <li> Heatshock at 42°C for 90 seconds | ||
+ | <li> Incubation on ice for 2 minutes | ||
+ | <li> Addition of 500µl SOC-Medium | ||
+ | <li> Incubation at 37°C for 60 minutes | ||
+ | <li> 100µl of suspension wase plated on agarplates containing ampicilin | ||
+ | <li> Incubation at 37° C over night | ||
+ | </ul> | ||
+ | </ol> | ||
+ | |||
+ | <h2> | ||
+ | Day 2, 15/06/26: | ||
+ | </h2> | ||
+ | |||
+ | <h3> | ||
+ | 1. Picking colonies for overnight cultures (ONK)</h3> | ||
+ | |||
+ | <ul> | ||
+ | |||
+ | <li> Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000) | ||
+ | <li> Afterwards incubation overnight at 37°C | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <h2> | ||
+ | Day 3, 15/06/29: | ||
+ | </h2> | ||
+ | |||
+ | <h3> | ||
+ | 1. DNA-preparation via alkaline Lysis | ||
+ | </h3> | ||
+ | |||
+ | <ul> | ||
+ | <li> Experimental procedure according to " (")Protocol 1: Alkaline Lysis " (") | ||
+ | <li> Changes to protocol: | ||
+ | → No centrifugation of ONK | ||
+ | → Two 2ml reaction tubes filled with ONK instead | ||
+ | </ul> | ||
+ | |||
+ | <h3> 2. Picking of yeast colonies </h3> | ||
+ | |||
+ | <ul> | ||
+ | <li> Two yeast colonies picked out of YPED<li>Medium plates from Day 1 (see day 1/1.) | ||
+ | <li> Clone 1 named A | ||
+ | <li> Clone 2 named B | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <h2> | ||
+ | Day 4, 15/06/30: | ||
+ | </h2> | ||
+ | |||
+ | |||
+ | <h3> | ||
+ | 1. Restriction digest of the obtained DNA-Solutions | ||
+ | <h3> | ||
+ | <li> Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table | ||
+ | |||
+ | <table> | ||
+ | <tr> <th>Reagent</th> <th>Volume for one sample </th> <th>Mastermix for four samples </th> </tr> | ||
+ | <tr><th>EcoRI </th> <th>0.5 µl </th> <th>2 µl</th> </tr> | ||
+ | <tr><th>EcoRV </th> <th>0.5 µl </th> <th>2 µl</th> </tr> | ||
+ | <tr><th>Tango Buffer </th> <th>4 µl </th> <th>16 µl</th> </tr> | ||
+ | <tr><th>Add 20µl ddH2O </th> <th>14 µl </th> <th>56 µl</th> </tr> | ||
+ | <tr><th>&Sigma <th> </th> <th>19 µl </th> <th>76 µl</th> </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <li> 19 µl out of the mastermix were transferred to three 1.5 ml reaction tubes | ||
+ | <li> 1 µl YCplac22/YEplac181/YIplac204 solution obtained in 3.1 (see: p. 2) were given in one mastermix tube each | ||
+ | <li> 1µl K80100 solution obtained in 3.1 (see: p. 2) was added to following reagents: | ||
+ | |||
+ | Reagent Volume for one sample | ||
+ | PstI 0.5 µl | ||
+ | Buffer O 2 µl | ||
+ | Add 20µl ddH2O 16.5 µl | ||
+ | Sum 19 µl | ||
+ | |||
+ | <li> Digests were incubated at 37°C for 3 h | ||
+ | |||
+ | |||
+ | \subtitle 2. Gelelectrophoresis of digested DNA | ||
+ | |||
+ | <li> Gelectrophoresis was executed according to protocol 2 | ||
+ | <li> 2 µl 6x staining solution were given unto 10 µl digest | ||
+ | <li> Samples were loaded onto the gel according to following scheme | ||
+ | => DNA<li>1kb<li>Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\DNA<li>1kb<li>Marker (6µl) | ||
+ | <li> Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes | ||
+ | |||
+ | hier Bilder 150630a <li>c einfügen (http://www.studon.uni<li>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<li>Description as well as cropping. | ||
+ | |||
+ | Figure 2: Gelphoto after 1h 40 minutes. | ||
+ | |||
+ | Figure 3: Gelphoto after 2h 20 minutes. | ||
+ | |||
+ | |||
+ | |||
+ | Day 5, 15/07/01: | ||
+ | |||
+ | |||
+ | \subtitle 1. Repetition of restriction digest for Ycplac 204 and K801000 digested | ||
+ | |||
+ | |||
+ | |||
+ | <li>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 | ||
+ | Sum 18 µl 54 µl | ||
+ | |||
+ | |||
+ | <li> 2µl of obtained DNA<li>Solution were transfered to 18 µl of mastermix | ||
+ | <li> Incubation at 37°C for 3 hours | ||
+ | <li> Gelelectrophoresis was conducted according to protocol 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 | ||
+ | |||
+ | => 6µl DNA<li>1KB<li>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 DNA<li>1KB<li>MARKER | ||
+ | |||
+ | Bild 150701a aus http://www.studon.uni<li>erlangen.de/studon/goto.php?target=fold_1325854 | ||
+ | Figure 4: Photo of gelelectrophoresis at 120 V after 50 minutes. | ||
+ | |||
+ | |||
+ | \subtitle 2. Preparation of S. Cerevisiae tryptophan negative plates | ||
+ | <li> Added following substances in two 1 liter flasks: | ||
+ | |||
+ | <li> 3.35g Difco yeast nitrogen base 2/o aminoacid | ||
+ | <li> 5.5 g CAA vitamin assay | ||
+ | <li> 10 g Glucose | ||
+ | <li> 83.0 mg Tyrosin<li>Uracil<li>Adenin<li>mix | ||
+ | <li> 50.5 mg Leucin | ||
+ | <li> 22g Agar | ||
+ | |||
+ | |||
+ | \subtitle 3. Preparation of S. Cerevisiae full media plates | ||
+ | <li>Added following substances in two 1 liter flasks | ||
+ | <li>5.3g yeast extract | ||
+ | <li>11g Bacopepton | ||
+ | <li>10g Glucose | ||
+ | <li>22.7 mg Adenin | ||
+ | <li> 11g Agar | ||
+ | |||
+ | \subtitle 4. Overnightculture of YIplac204 positive bacteria | ||
+ | |||
+ | <li> two times 5 ml ampicilin medium (80µg/ml) with 2 amp<li>ressistant bacteria picked of plate from 1.2 inoculated | ||
+ | <li> Incubation at 37°C over night | ||
+ | |||
+ | |||
+ | |||
+ | Day 6, 15/07/02: | ||
+ | |||
+ | \subtitle 1. Moulding of Agar<li>plates | ||
+ | <li> Added 500ml destilled Water to each Flask of 5.2 and 5.3 | ||
+ | <li> Short mixing with stirring bar | ||
+ | <li> Flask autoclaved | ||
+ | <li> Stirring with stirring bar for 20 minutes at room temperature | ||
+ | <li> Moulding of plates underneath a laminar flow hood | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subtitle 2. DNA<li>Preperation of overnight culture (see 5.4. p.:) | ||
+ | <li> Conducted analogous to Day 3.1. according to protocol 1 | ||
+ | |||
+ | |||
+ | |||
+ | \subtitle 3. Digest of obtained DNA | ||
+ | |||
+ | |||
+ | <li> 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 6µl | ||
+ | Add 20µl H2O 15.5µl 46.5µl | ||
+ | Sum 18 µl 18 µl | ||
+ | |||
+ | <li> 2 µl DNA DNA<li>preperation of each dublicat of YIp204 were added onto 18µl mastermix | ||
+ | <li> Incubated for 3 hours at 37°C | ||
+ | <li> Electrophoresis was conducted according to protocol 2 | ||
+ | <li> Added 4µl 6x staining buffer to each digest after incubation time | ||
+ | <li> Added 4µl 6x staining buffer to 20µl undigest DNA<li> Preperation | ||
+ | <li> Loaded gel according to following scheme | ||
+ | => 6µl DNA<li>1KB<li>MARKER \\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested | ||
+ | <li> DNA<li>fragments of unknown origin were found | ||
+ | |||
+ | 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= | ||
+ | |||
+ | |||
+ | \subtitle 4. Overnight Culture of Strains 4196 and K699 for transformation | ||
+ | <li> 3 ml YEPD Medium was given to each of 4 reaction tubes | ||
+ | <li> Two yeast colonies of 4196 and K669 were picked | ||
+ | <li> Incubation at 30°C over night | ||
+ | |||
+ | |||
+ | \subtitle 5. Restriction digest of Vector DNA for the transformation | ||
+ | <li> 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 | ||
+ | Sum 40 µl | ||
+ | |||
+ | <li> Incubation over night at 37°C | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subtitle6. Overnight culture of YIplac204 | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | Day 7, 15/07/03 | ||
+ | |||
+ | |||
+ | \subtitle 1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22 and Ylplac204 | ||
+ | |||
+ | <li> over night culture diluted 1:100 with MQ (10 µl suspension and 990 µl MQ) | ||
+ | <li> measurement 0D600: | ||
+ | 699 A = 0.203 | ||
+ | 699 B = 0.143 <li>> used | ||
+ | 7196 A = 0.12 | ||
+ | 4196 B = 0.139 <li>> used | ||
+ | <li> two flasks filled with YEPG next to Bunsenburner | ||
+ | A: 50 ml | ||
+ | B: 25 ml <li>> Media (wrong estimate) | ||
+ | <li> in flask A 2 ml suspension 699 oD600 = 0.30 | ||
+ | <li> in flask B 1 ml suspension 4196 oD600 = 0.293 | ||
+ | <li> 3 h at 30 °C and incubated while shaking | ||
+ | |||
+ | \subtitle 2. DNA<li>Preperation with YIplac204 overnight culture (see 6.6) | ||
+ | <li> Experiment conducted according to protocol 1 | ||
+ | |||
+ | |||
+ | \subtitle 3. Gelelectrophoresis of overnight digestion and DNA Preparation | ||
+ | |||
+ | <li> Experiment conducted according to protocol 2 | ||
+ | <li> hanges to protocol: | ||
+ | <li>> 1.2 % agarose gel | ||
+ | <li> 5 µl of overnight digest added to 1 µl 6x staining buffer | ||
+ | <li> loaded onto gel according to following scheme | ||
+ | => 6 µl 1kb<li>DNA<li>marker \\ overnight digest | ||
+ | |||
+ | |||
+ | Hier 150703a einfügen ort | ||
+ | |||
+ | http://www.studon.uni<li>erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= | ||
+ | |||
+ | Beschriftung | ||
+ | Figure: Gelelectrophoresis of EcoRV digested vector YIplac204 (3545 bp). No contamination detected. DNA concentration estimated to be about 12 ng/µl. | ||
+ | |||
+ | |||
+ | <li> After the photo was taken | ||
+ | <li> DNA<li>Preperation from Day 7 2. ( see page 10) loaded on same gel | ||
+ | <li> Scheme : 6 µl 1kb<li>DNA<li>marker //overnight digest //empty// marker // Miniprep | ||
+ | |||
+ | Hier 150703b einfügen ort: | ||
+ | |||
+ | http://www.studon.uni<li>erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= | ||
+ | |||
+ | Figure: Gelelectrophoresis of preparated vector YIplac204 (3545 bp). No contamination detected. DNA concentration estimated to be over 70 ng/µl. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subtitle 5. Precipitation of plasmid<li>DNA of digested YIplac204 | ||
+ | |||
+ | <li> 3.5 µl 3M NaAC added to DNA solution | ||
+ | <li> 1.5 µl Glycogen (Thermo Scientific) added | ||
+ | <li> 100µl 95% Ethanol added | ||
+ | <li> Incubation for 5 minutes at room temperature | ||
+ | <li> Centrifugation (fullspeed, 5 min, room temperature) | ||
+ | <li> DNA appears as a small pellet | ||
+ | <li> Supernatant removed | ||
+ | <li> Pellet washed in two volumes (240 µl) EtOH 70 % | ||
+ | <li> Dried at room temperature for 30 min | ||
+ | <li> DNA solved in 5 µl TE | ||
+ | |||
+ | \subtitle 4. Transformation of the yeast K699 | ||
+ | <li> 25 ml yeast culture from the flask given in 50 ml falcon | ||
+ | <li> oD determined 699 = 0.76 | ||
+ | 4196 = 0.75 | ||
+ | <li> Centrifugation (5 min, 3500 rpm, room temperature) | ||
+ | <li> Supernatant 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 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 (4) transferred in 1.5ml ml reaction tubes | ||
+ | <li> Centrifugated for 10 s and supernatant discarded | ||
+ | <li> All samples of 4196 discarded (too few DNA) | ||
+ | <li> Mix for transformation added (adhered to order as written below) | ||
+ | <li>> 240 µl 50 % PEG 3350 | ||
+ | <li>> 36 µl 1 M LiAc | ||
+ | <li>> 5 µl carrier DNA (10mg/ml) | ||
+ | <li>> 4 µl YEP122 (positive control) / 5 µl YIP204/ 3µl YCplac22/ 5µl ddH2O (negative control) | ||
+ | <li>> 65 µl of ddH2O to YEplac122 <li>> 360 µl | ||
+ | 64 µl of ddH2O to YIplac204 <li>> 360 µl | ||
+ | 66 µl ddH2O to YCplac22 <li>> 360 µl | ||
+ | 64 µl ddH2O to negative control <li>> 360 µl | ||
+ | <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, pellet resuspended in 400 µl H2O | ||
+ | <li>> YEplac122 200 µl plated | ||
+ | <li>> YIplac204 400 µl plated | ||
+ | <li>> YCplac22 200 µl plated | ||
+ | <li>> negative control 200 µl plated | ||
+ | <li> incubated (72 h, 30 °C) | ||
+ | |||
+ | |||
+ | |||
+ | Day 8, 15/07/06: | ||
+ | |||
+ | \subtitle 1. Examination of 7 4. | ||
+ | <li> Colonies grown! | ||
+ | <li> Transformation works | ||
+ | |||
+ | \subtitle 2. Resuspension of IDT geneBricks his_spacer adh1 and his_rep_klein | ||
+ | <li> 500 ng in 50 µl TE given (c = 10 ng/ml) | ||
+ | <li> incubated (50 °C, 20 min) | ||
+ | <li> vortexed and centrifugated (10 s at fullspeed) | ||
+ | |||
+ | |||
+ | \subtitle 3. Restriction digest for ligation of his_spacer adh1 and his_rep_klein | ||
+ | |||
+ | <li> calculations for needed amount of DNA via following formula | ||
+ | |||
+ | m(plasmid) * lengt (insert)/length(Vector)*5 <li>> 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 | ||
+ | |||
+ | |||
+ | <li> following restriction digests were constructed: | ||
+ | insert his_spacer_adh1/ insert His_Rep_klein | ||
+ | |||
+ | DNA= 40 µl MM for 3 samples | ||
+ | EcoRI 2 µl 6 µl | ||
+ | PstI 2 µl 6 µl | ||
+ | Buffer0 20 µl 60 µl | ||
+ | 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 | ||
+ | Buffer0 5 µl 20 µl | ||
+ | add 50µl ddH2O 37µl 148 µl | ||
+ | |||
+ | Vector K801000 | ||
+ | DNA 40 µl | ||
+ | RNAse 1 µl | ||
+ | EcoRI 2 µl | ||
+ | PstI 2 µl | ||
+ | Buffer0 10 µl | ||
+ | add100 ddH2O 45 µl | ||
+ | |||
+ | <li> large volumes were chosen because of the high EDTA<li>concentration | ||
+ | <li> over night incubation at 37 °C | ||
+ | |||
+ | 4. Speedjet PCR<li>cloning with his3_rep_klein and his_spacer_adh1 | ||
+ | |||
+ | <li> as a backup the following speedjet Samples were generated | ||
+ | 2 µl 10 x ligase buffer | ||
+ | 2.5 µl DNA solution | ||
+ | 1 µl vector | ||
+ | add 19 µl ddH2O <li>> 13.5µl | ||
+ | 1 µl T4Ligase | ||
+ | Incubation (ca. 10 min) | ||
+ | |||
+ | |||
+ | |||
+ | 5. Transformation | ||
+ | |||
+ | <li> DH5alpha<li>E.colis unfreezed | ||
+ | <li> Each 5 µl from Day8 4. given on top of 50 µl competent cells | ||
+ | <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 µl E. coli | ||
+ | <li> incubate for about 15 min on ice | ||
+ | <li> heat shock for 90 s | ||
+ | <li> 2 min on ice | ||
+ | <li> 500 µl SOC medium added | ||
+ | <li> incubated (45 min, 37 °C) | ||
+ | <li> centrifugation (2 min, 7000 rpm) | ||
+ | <li> remove 450 µl supernatant | ||
+ | <li> E.coli in remained 100 µl resuspended | ||
+ | <li> 100 µl plated on ampiciline plates | ||
+ | |||
+ | Day 9, 15/07/07 | ||
+ | |||
+ | \subtitle 1. Analysis of over night digest from Day 8 3. | ||
+ | |||
+ | <li> Electrophoresis was conducted according to protocol 2 | ||
+ | <li> The gel was loaded with 10 % digest volume | ||
+ | <li> 6x staining buffer was added according to volume (given in Brackets) | ||
+ | |||
+ | => | ||
+ | YEplac181/ YCplac22/ YIplac204 <li>> 5 µl (1 µl ) | ||
+ | K801000 <li>> 10 µl (2 µl ) | ||
+ | Inserts(his3_rep_klein and his_spacer_adh1) <li>> 20 µl (4 µl ) | ||
+ | |||
+ | <li> Pockets were loaded according to following scheme | ||
+ | |||
+ | => 1 kb DNA<li>marker //YEplac181//YCplac22//YIplac204//K801000//his_spacer_adh1//his3_rep_klein | ||
+ | |||
+ | hier Bild 150707a einfügen Ordner | ||
+ | |||
+ | |||
+ | figure: Linearisation of all vectors achieved. DNA<li>concentration of Reporter Insert his3_rep_klein below detection limit. Insert his_spacer_adh1 only just above the detection range. | ||
+ | |||
+ | |||
+ | \subtitle 2. Restriction digestion 204 | ||
+ | |||
+ | <li> 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 | ||
+ | Sum 200 µl | ||
+ | <li> incubation (2h, 37 °C) | ||
+ | |||
+ | |||
+ | |||
+ | \subtitle 3. Gelelectrophoresis for extraction | ||
+ | <li> Gelelectrophoresis conducted according to protocol 2 | ||
+ | <li> Middlepocket of gel loaded with 45 µl YCplac22 Day 8 3.according to following scheme: | ||
+ | 6µl 1 kb DNA<li>marker // empty // YCplac22 // empty | ||
+ | |||
+ | <li> Changes to protocol: | ||
+ | <li>> large comb used | ||
+ | <li>> run for 2h at 50V | ||
+ | |||
+ | \subsection 4. Gelextraction | ||
+ | |||
+ | <li> Gel fragment weighted: 470 mg | ||
+ | <li> 3 times the volume QC<li>buffer added <li>> 1410 µl QC<li>buffer added | ||
+ | <li> 10 min at 50 °C gel dissolved | ||
+ | <li> After 3 min and 7 min for 5<li>7 s vortexed | ||
+ | <li> 450 µl isopropanol added | ||
+ | <li> Sample inverted and shortly vortexed | ||
+ | <li> 800 µl given on speedcolumn with wastetube | ||
+ | <li> Centrifugation (13300 rpm, 1 min) <li>> flowthrough discarded | ||
+ | <li> Step 3 times repeated | ||
+ | <li> 0,75 ml PE buffer added on column for cleaning | ||
+ | <li> Centrifugation (13300 rpm, 1 min) | ||
+ | <li> Flowthrough discarded | ||
+ | <li> Centrifugation (13300 rpm, 1 min) | ||
+ | <li> Flowthrough discarded | ||
+ | <li> Column transferred on 1.5 ml tube | ||
+ | <li> 30 µl Elutionbuffer added on column | ||
+ | <li> Centrifugation (13300 rpm, 1 min) | ||
+ | <li> Eluate used for further experiments | ||
+ | |||
+ | |||
+ | \subsection 5. Further cleaning with PCR<li>Purification<li>Kit | ||
+ | |||
+ | <li> 180 µl sample given to 900 µl PB buffer given | ||
+ | <li> 800 µl Sample given on column | ||
+ | <li> column centrifugated (13300 rpm, 1 min) | ||
+ | <li> remaining 280 µl given on column | ||
+ | <li> column centrifugated at 13300 rpm | ||
+ | <li> both times flowthrough was discarded | ||
+ | <li> column loaded with 750 µl PE | ||
+ | <li> centrifugation (13300 rpm, 1 min) | ||
+ | <li> flowthrough discarded | ||
+ | <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 | ||
+ | |||
+ | \subsection 6. Controlgelelectrophoresis of eluate and digestion of YIplac204 and YCplac22 Day 9 2. | ||
+ | |||
+ | <li> Gelelectrophoresis according to protocol 2 | ||
+ | <li> Gel loaded with samples according to following scheme: | ||
+ | Standard // YCplac22 //YIplac204 digested // empty // Standard // Insert his3_reporter_klein | ||
+ | 6 µl 3 µl 20 µl 6 µl 2,8 µl | ||
+ | |||
+ | <li>Changes to protocol: | ||
+ | <li>> Gelelectrophoresis for 45 min | ||
+ | <li>> note: insert was added after 20 min | ||
+ | |||
+ | Hier Bild 150707b aus dem Gelphoto ordner | ||
+ | |||
+ | |||
+ | |||
+ | Figure: Controlgel after purification. Vector DNA<li>concentration is still above the 25ng per µl. Detection of the Insert his_rep_klein was possible. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subsection 7. Overnight culture of Pjet<li>transformed E. coli | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | Day 10, 15/07/08: | ||
+ | |||
+ | \subsection 1. Ligation of the linearised vector YCplac22 and the his3_rep_klein | ||
+ | |||
+ | <li> 3 samples for ligation generated | ||
+ | |||
+ | <li>> ligation with insert | ||
+ | |||
+ | <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 | ||
+ | |||
+ | <li>> Re<li>Ligation control without insert | ||
+ | |||
+ | <li> 3 µl vector (YCPlac22) linearized | ||
+ | <li>14 µl ddH2O | ||
+ | <li> 2 µl ligase buffer | ||
+ | <li> 1 µl T4 Ligase | ||
+ | |||
+ | <li>> control for complete digestion | ||
+ | |||
+ | <li> 3 µl vector (YCPlac22) linearized | ||
+ | <li> 15 µl H2O | ||
+ | <li> 2 µl ligase buffer | ||
+ | <li> ligation incubated for 3 h at room temperature | ||
+ | |||
+ | \subsection 2. Miniprep from overnight culture Day 9 7. | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | \subsection 3. Digestion of Miniprep | ||
+ | |||
+ | <li>Digest prepared | ||
+ | <li>> 1 µl DNA from the miniprep (see p.:19:) <li>> 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 | ||
+ | |||
+ | <li> digestion for 3 h at 37 °C | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subsection 4. Mouding of Chloramphenicol agar plates | ||
+ | |||
+ | <li>TY<li> 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 | ||
+ | |||
+ | \subsection 5 Preparation of X<li>gal plates | ||
+ | <li> 40µl 2% x<li>gal spread on 6 ampicilin enriched plates | ||
+ | <li> 40µl IPTG spread on the dried plates | ||
+ | <li> 40µl dH2O spread on the dried plates | ||
+ | |||
+ | \subsection 6 Transformation of Ligation and EYFP (BBa_E2030) | ||
+ | <li> Each of folowing DNA<li>samples added to 50 µl DH5alpha on ice | ||
+ | <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) | ||
+ | |||
+ | <li>Transformation analogous to 8.5 | ||
+ | <li>Transformed \textit{E. coli} samples spread on ampiciline plates as followed | ||
+ | |||
+ | <li>> 100 µl ligation | ||
+ | <li>> 200 µl ligation | ||
+ | <li>> 200 µl ligase + vector | ||
+ | <li>> 200 µl pBluescript | ||
+ | <li>> 200 µl H2O | ||
+ | |||
+ | <li>Remaining transformed bacteria spread on chloramphenicole agar plates | ||
+ | |||
+ | <li>> 200 µl EYFP (BBa_E2030) | ||
+ | <li>> 200 µl H2O | ||
+ | |||
+ | \subsection 7. Gelelectrophoresis of the Digestion from 3. (see above) | ||
+ | <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: | ||
+ | |||
+ | his3_rep_klein A // his3_rep_klein B // his_spacer_adh1 A // his_spacer_adh1 B// YIplac204 A //YIplac 204 B// 1kb DNA<li>marker | ||
+ | |||
+ | Foto: 150708a.png | ||
+ | http://www.studon.uni<li>erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= | ||
+ | |||
+ | |||
+ | |||
+ | Bildunterschrift. Gelfoto of pjet digest and control of YIP204 digest. | ||
+ | |||
+ | |||
+ | Day 11, 15/07/09 | ||
+ | |||
+ | 1. Cultures picked for over night culture | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | Day 12, 15/07/10 | ||
+ | |||
+ | \subsection 1. Miniprep of the over night culture from Day 11 1. (see above) | ||
+ | |||
+ | <li> conducted according to protocol 1 | ||
+ | <li> eluted in 30µl 1x TE | ||
+ | |||
+ | |||
+ | \subsection 2. Restriction digestion | ||
+ | <li> Restriction for all DNA<li>Preparation (14) | ||
+ | |||
+ | <li>> DNA Solution 3 µl MM for 15 | ||
+ | |||
+ | EcoRI 0.5 µl 7.5 µl PstI 0.5 µl 7.5 µl | ||
+ | Buffer O 2 µl 30 µl | ||
+ | ddH2O 14 µl 210 µl | ||
+ | |||
+ | <li> incubation (1.5 h, 37 °C) | ||
+ | |||
+ | \subsection 3. Gelelectrophoresis | ||
+ | |||
+ | <li> Gelelectrophoresis performed according to protocol 2 | ||
+ | <li> Changes to protocol: | ||
+ | |||
+ | <li>> Volume for two 1% agarosegels produced 170 ml (100 ml+70 ml) | ||
+ | <li>> 13,6 µl Roth Ethidiumbromide added | ||
+ | |||
+ | <li> Samples from Day 12 2. (see above) were added to 4 µl staining solution | ||
+ | <li> Gels were loaded as followed: | ||
+ | |||
+ | <li>> Gel I: 1kb DNA<li>marker // YCPlac22 + insert 1 // | ||
+ | 2 // | ||
+ | 3 // | ||
+ | 4 // | ||
+ | 5 // | ||
+ | 6 // | ||
+ | 7 // | ||
+ | 8 // | ||
+ | |||
+ | <li>> Gel II: 1kb DNA<li>marker // YCPlac22 + insert 9 // | ||
+ | 10 // | ||
+ | 11 // | ||
+ | 12 // | ||
+ | empty // | ||
+ | YFP clone 1 // | ||
+ | YFP clone 2 // | ||
+ | stamdard // | ||
+ | |||
+ | |||
+ | |||
+ | <li> Results not expected ratio insert vector seems tilted | ||
+ | <li> Over night culture of all samples analogous to Day 11 1. | ||
+ | |||
+ | <li>Bilder einfügen 150710a und 150710b | ||
+ | |||
+ | Bildunterschriften | ||
+ | a) Gelphoto of gel I. Expected sizes for the clones are 4854 bp, 750 bp and 5604 bp | ||
+ | b) Gelphoto of gel II. Expected sizes for the clones are 4854 bp, 750 bp and 5604 bp. For YFP 778 bp 2073 bp and 2851 bp. | ||
+ | |||
+ | |||
+ | Day 13, 15/07/11 | ||
+ | |||
+ | \subsection 1. Miniprep via Quiagen column | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | \subsection 2. Restriction digestion | ||
+ | <li> DNA<li>Solutions obtained in Day 13 1. (see above) EcoRI PstI digested | ||
+ | |||
+ | <li> Mastermix created | ||
+ | |||
+ | <li>> single sample Mastermix for 13 Samples | ||
+ | 1 µl DNA <li> | ||
+ | 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 | ||
+ | |||
+ | <li> incubation (2 h, 37 °C) | ||
+ | |||
+ | \subsection 3. Gelelectrophoresis | ||
+ | |||
+ | <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 | ||
+ | |||
+ | 1kb DNA<li>marker // | ||
+ | YCPlac 22 + insert 1 // | ||
+ | YCPlac 22 + insert 2 // | ||
+ | YCPlac 22 + insert 3 // | ||
+ | YCPlac 22 + insert 4 // | ||
+ | ... | ||
+ | YCPlac 22 + insert 12 // | ||
+ | |||
+ | Bild einfügen 150713a im Gelfoto ordner | ||
+ | |||
+ | Beschriftung Gelfoto of control digest. Expected sizes for the clones are 4854 bp, 750 bp and 5604 bp. | ||
+ | |||
+ | |||
+ | Day 14, 15/07/14 | ||
+ | |||
+ | \subsection 1. Restriction digestion XhoI, Sal I from YCPlac22 + insert10 (13/13) | ||
+ | |||
+ | <li> restriction digestion conducted | ||
+ | |||
+ | <li>> 20 µl DNA YCPlac22 + insert10 from day 13 | ||
+ | 2 µl Sal I | ||
+ | 4 µl XhoI | ||
+ | 10 µl Buffer0 | ||
+ | 64 µl ddH2O | ||
+ | |||
+ | <li> incubation (2 h, 37 °C) | ||
+ | |||
+ | \subsection 2. Addition of SalI and XhoI restriction sites via PCR | ||
+ | |||
+ | |||
+ | Sample water control | ||
+ | H2O 71 µl 73 µl | ||
+ | 5 x buffer 20 µl 20 µl | ||
+ | template 2 µl <li> | ||
+ | Forward Primer 2 µl 2 µl | ||
+ | Reverse Primer 2 µl 2 µl | ||
+ | Fusion Polymerase 1 µl 1 µl | ||
+ | |||
+ | Sum 100 µl 100 µl | ||
+ | |||
+ | |||
+ | <li> program: 1. 98.0 °C 15 s | ||
+ | 2. 98.0 °C 10 s repeated 30 times | ||
+ | 3. 72.0 °C 15 s | ||
+ | 4. 72.0 °C 3 s | ||
+ | |||
+ | |||
+ | \subsection 3. Gelelectrophoresis of the restriction digestion and PCR | ||
+ | |||
+ | <li> Gelelectrophoresis performed according to protocol 2 | ||
+ | <li> Changes to protocol | ||
+ | <li>> 70 ml 1 % agarose gel moulded | ||
+ | |||
+ | <li> 1 µl staining solution added to 5 µl water control | ||
+ | <li> 1 µl staining solution added to 5 µl PCR<li>sample | ||
+ | <li> 2 µl staining solution added to 10 µl of digest | ||
+ | <li> According to following scheme loaded | ||
+ | 1kb DNA<li>marker // digestion // water control // PCR | ||
+ | |||
+ | Bild einfügen 150714a im gelfoto ordner | ||
+ | |||
+ | Beschriftung: The digested vector (5604 bp) was free of contamination. The PCR of YFP was succesfull as a | ||
+ | DNA fragment of 788 is clearly to be seen and the water control shows no contamination | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | \subsection 4. Purification of the PCR fragment | ||
+ | |||
+ | <li> Analogous to Day 9 5. (p.:16) | ||
+ | <li> pellet was solved in 30 µl EB | ||
+ | |||
+ | |||
+ | \subsection 5. Restriction digestion of the YFP<li>PCR fragment | ||
+ | |||
+ | <li> purified insert digested with XhoI Sal I | ||
+ | |||
+ | <li>> 30 µl DNA | ||
+ | 2 µl Sal I | ||
+ | 4 µl XhoI | ||
+ | 10 µl Buffer0 | ||
+ | 54 µl ddH2O | ||
+ | |||
+ | <li> incubation (3 h, 37 °C)) | ||
+ | |||
+ | |||
+ | \subsection 6. Gelextraction of the digested vector | ||
+ | |||
+ | <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 | ||
+ | |||
+ | |||
+ | Day 15, 15/07/15 | ||
+ | |||
+ | \subsection 1. Purification of the digestion from 14/5 | ||
+ | |||
+ | <li> Analogue to Day 9 5. | ||
+ | <li> Changes to Day 9 5. | ||
+ | <li>> 380 µl PB used | ||
+ | <li>> eluted in 30 µl Elution Buffer | ||
+ | |||
+ | \subsection 2. Dephosphorylation of the vector | ||
+ | |||
+ | <li> alkaline phosphatase mix created | ||
+ | |||
+ | <li>> 20 µl TCPlac22 + insert XhoI Sal I digested | ||
+ | 3 µl FAP buffer | ||
+ | 1 µl FAP | ||
+ | 6 µl H2O | ||
+ | |||
+ | <li> incubation (20 min, 37 °C) | ||
+ | <li> incubation (5 min, 75 °C) | ||
+ | |||
+ | |||
+ | \subsection 3. Ligation YCPlac22 + insert his3_rep_klein and YFP | ||
+ | |||
+ | <li> 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 | ||
+ | |||
+ | <li> incubation (3 h, room temperature) | ||
+ | |||
+ | |||
+ | \subsection 4. Transformation of the ligation | ||
+ | |||
+ | <li> performed analogue to Day 8 5. | ||
+ | <li> transformation plated as followed: | ||
+ | |||
+ | <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<li>Bluescript and H2O on ampiciline plated | ||
+ | |||
+ | |||
+ | |||
+ | Day 16, 15/07/16 | ||
+ | |||
+ | \subsection 1. Picking of colonies | ||
+ | <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 | ||
+ | |||
+ | \subsection 2. Retransformation of the ligation | ||
+ | |||
+ | <li> Repition of Day 15 3. (see above) | ||
+ | |||
+ | |||
+ | Day 17, 15/07/17 | ||
+ | |||
+ | \subsection 1. Miniprep of the over night culture | ||
+ | |||
+ | <li> Performed according to protocol 1 | ||
+ | |||
+ | \subsection2. Restriction digestion from the Miniprep | ||
+ | |||
+ | <li> restriction digest created | ||
+ | |||
+ | 1 µl Miniprep Mastermix for 21 samples | ||
+ | |||
+ | 0.5 µl Sal I 10.5 µl Sal I | ||
+ | 1 µl XhoI 21 µl XhoI | ||
+ | 2 µl Buffer0 42 µl Buffer0 | ||
+ | 15.5 µl ddH2O 325.5 µl ddH2O | ||
+ | |||
+ | <li> digestion (2 h, 37 °C) | ||
+ | |||
+ | \subsection 3. Gelelectrophoresis | ||
+ | <li> Gelelectrophoresis performed according to protocol 2 | ||
+ | <li> 4 µl staining solution was added to each digest | ||
+ | <li> 6 µl 1kb DNA<li>marker loaded | ||
+ | <li> scheme: 1kb DNA<li>marker // Miniprep 1 <li> 10 = Gel I | ||
+ | 1kb DNA<li>marker // Miniprep 11 <li> 20 = Gel II | ||
+ | <li> expected fragments ~ 5000 bp | ||
+ | ~ 700 bp | ||
+ | |||
+ | Bilder einfügen 150717a und 150717b | ||
+ | |||
+ | Beschriftung | ||
+ | a)GelI. Restriction sites XhoI and SalI are compatible, but after XhoI<li>SalI<li>ligation the restriction site is removed. Thus "homo"<li>ligation creates an insert after XhoI<li>SalI digestion whereas "hetero"<li>ligation does not. | ||
+ | b)GelII Restriction sites XhoI and SalI are compatible, but after XhoI<li>SalI<li>ligation the restriction site is removed. Thus "homo"<li>ligation creates an insert after XhoI<li>SalI digestion whereas "hetero"<li>ligation does not. | ||
+ | |||
+ | <li> 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 | ||
+ | <li> Two colonies picked of each strain | ||
+ | <li> Inoculation of 5 ml YEPD<li>Medium each | ||
+ | <li> Incubation overnight at 28°C | ||
+ | |||
+ | Day 19, 15/07/21 | ||
+ | 1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22<li>YFP | ||
+ | <li> over night culture dilitued 1:100 with MQ (10 µl suspension and 990 µl MQ) | ||
+ | <li> measurement 0D600: | ||
+ | 699 A = 0.019 <li>> used | ||
+ | 699 B = 0.014 | ||
+ | 7196 A = 0.108 <li>> used | ||
+ | 4196 B = 0.056 | ||
+ | <li> two flasks filled with YEPG next to Bunsenburner | ||
+ | A: 25 ml | ||
+ | B: 25 ml | ||
+ | |||
+ | <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 <li>> 699 = 0.543 | ||
+ | 4196 = 0.503 | ||
+ | <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) | ||
+ | <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<li>YFP 1, 8, 9, 12, 14 and 18 | ||
+ | <li>> 64 µl of ddH2O | ||
+ | <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) | ||
+ | |||
+ | Day 20, 15/07/24 | ||
+ | 1. Convokal Microskopy | ||
+ | <li> One colony per construct picked and diluted in Water | ||
+ | <li> Microscopy <li>> see result section | ||
+ | |||
+ | |||
+ | |||
+ | |||
<p>1 von Adrian</p> | <p>1 von Adrian</p> | ||
<p>1 von Filip</p> | <p>1 von Filip</p> |
Revision as of 07:57, 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
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
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 | |
&Sigma | 19 µl | 76 µl |
1 von Adrian
1 von Filip
1 von Vlady (schon auf Studon)
1 von Frederike