Labprotocol: S. cerevisiae GFP

Inhaltsverzeichnis Day 1: 3 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of strain collection 3 2. Transformation of Vectors YCplac22, Yeplac 181 and YIplac204 in DH5α E. coli 3 Day 2: 3 1. Picking colonies for overnight cultures 3 Day 3: 3 1. Preperation via alkaline Lysis 3 Day 4: 4 1. Restriction digest of the obtained DNA-Solutions 4 2. Gelelectrophoresis of digested DNA 5 Day 5, 01.07.15: 7 1. Repetition of restriction digest for Ycplac 204 and K801000digested 7 2. Preperation of S. Cerevisiae tryptophan negative plates 8 3. Preparation of S. Cerevisiae full media plates 8 4. Overnightculture of YIplac204 positive bacteria 9 Day 6, 02.07.15 9 1. Moulding of Agar-plates 9 2. DNA-Preperation of overnight culture (see 5.4. p.:7) 9 3. Controldigestion of obtained DNA 9 4. Overnight Culture of Strains 4196 and K699 for transformation 10 5. Restriction digest of Vector DNA for the transformation 10 6. Overnight culture of YIplac204 11 Day 7, 03.07.15 11 1. Transformation with KCPlac22 and KlPlac202 ????? 11 2. Miniprep with KCPlac204 11 3. Gelelectrophoresis 12 4. Transfrmation of the yeast …... G99 12 5. Precipitation of plasmid-DNA ???????????? 13 Day 8, 06.07.15 13 1. Examination V7/4 13 2. Resuspension of IDT geneBricks xxxxxxxxxxxxx and xxxxxxxxxx 13 3. Restriction diggestion for ligation of xxxxxxxxxxxx and xxxxxxxxx 14 4. Speedjet PCR-cloning with cccccccccc and xxxxxxxxxxx 15 5. Transformation 15 Day 9, 07.07.15 15 1. Analysis of over night ?????? from 8/3 16 2. Restriction digestion 204 16 3. Gels for extraction 16 4. Gelextraction 16 5. Insertcleaning with PcR-Purification-Kit 17 6. Controlgel of eluate and digestion of 204 9/2 17 7. ÜNR 17 Day 10, 08.07.15 17 1. Ligation of the linearised vector VCPlac22 xxxxxxxxxxxxxxx 17 2. Miniprep from (9/7 S 20) 18 3. Digestion of Miniprep 18 7. Gelelectrophoresis of the Digestion from 3 19 Day 11, 09.07.15 19 1. Culture picking for over night culture 19 Day 12, 10.07.15 19 1. Miniprep of the over night culture from 11/1 19 2. Restriction digestion 19 3. Gelelectrophoresis 20 Day 1: 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of 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 DH5α 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: 1. Picking colonies for overnight cultures - Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000) - Incubation in 5ml ampicilin enriched medium (LB with 80 µg/µl) for 2 days at 8°C - Afterwards incubation overnight at 37°C Day 3: 1. Preperation via alkaline Lysis - Vortexing ÜNK (see 2.1./ p.: 2) - Transferring 2 ml out of ÜNK in two 2ml reaction tubes thus creating a dublicate - Centrifugation at 7000 rpm for 5 minutes (Centrifuge used: Haereus pico17; producer Thermo Fischer Scientific) - Removed supernatant - Pellets resuspended in 90µl GTE-Buffer (50 mM Glucose, 10 mM EDTA, 25 mM Tris/HCl pH8,0) - Vortexing for ca. 10 seconds - Addition of 180µl NaOH/SDS (1% SDS, 0,2 M NaOH) - Inverting tubes 4 times - Addition of 135µl Kac/Hac (3M KAc/2M HAc) - Inverting 10 times - Incubation for 5 minutes on ice - Centrifugation at 13,300 rpm for 15 minutes - Each supernatant transferred in one 1,5 reaction tube - Addition of 1 ml EtOH (100%) - Vortexing for 10 seconds - Incubation at 13,300 rpm for 10 minutes - Removal of ethanol - Addition of 1ml EtOH (70%) - Centrifugation at 13,300 rpm for 10 seconds - Repitition of the last two steps - Evaporation of remaining EtOH at room temperature - Resuspending pellet in 40µl TE-Puffer - Combining both duplicats of one sample Day 4: 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 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 H2O 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 3.1; p: 2 were given in one mastermix tube each - 1µl K80100 solution obtained in 3.1; p.:2 was added to following reagents: Reagent Volume for one sample Pst I 0,5 µl Buffer O 2 µl Add 20µl H2O 16,5 µl Σ 19 µl - Digest were incubated at 37°C for 3 h 2. Gelelectrophoresis of digested DNA - 2 µl 6x staining solution were given unto 10 µl digest - 9µl H20 were given unto 1 µl DNA solution obtained in 3.1; p.:2 each and 2 µl 6x staining solution were added - 1% agarose-gel was prepared by adding 1g agarose to 100 ml 1xTAE and heating in microwave oven - 8µl Ethidium Bromid were added after cooling of solution - Samples were added onto the gel according to following scheme => DNA-1kb-Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\DNA-1kb-Marker (6µl) - Gelelectrophoresis was conducted at 120 V - Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes Day 5, 01.07.15: 1. Repetition of restriction digest for Ycplac 204 and K801000digested -Mastermix created for K801000 and YCplac204 Reagent Volume for one sample Mastermix for three samples EcoRI 0,5 µl 1,5 µl EcoRV 0,5 µl 1,5 µl Tango Buffer 4 µl 12 µl Add 20µl H2O 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 - 100 ml 1% agarose gel were prepared in microwave oven - After light cooling 8µl Ethidium Bromid were added - 2 µl of 6x staining solution were added onto 10 µl of digest - Samples were loaded in pockets according to following scheme => 6µl DNA-1KB-MARKER\\ YIplac204 digested (01.07.15) \\ K801000 digested(01.07.15)\\ K801000(30.06.15)\\ YIplac204 (30.06.15)\\empty\\YEplac181 (30.06.15)\\empty\\ 6µl DNA-1KB-MARKER - Gelelectrophoresis at 120V for 50 minuts 2. Preperation 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) with 2 amp-ressistant bacteria picked of plate from 1.2 pg 2 inoculated - Incubation at 37°C over night Day 6, 02.07.15 1. Moulding of Agar-plates - Added 500ml destilled Water to each Flask of 5.2 and 5.3 p.:7 - 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-Preperation of overnight culture (see 5.4. p.:7) - Conducted analogous to 3.1. (see p.: 3) with a dublicat 3. Controldigestion of obtained DNA - Mastermix created to digest 2 µl DNA solution Reagent Volume for one sample Mastermix for three samples EcoRV 0,5 µl 1,5 µl Buffer R 2 µl 6 µl Add 20µl H2O 16,5 µl 46,5 µl Σ 18 µl 54 µl - 2 µl DNA DNA-preperation dublicat of YIp204 were added onto 18µl mastermix - Incubated for 3 hours at 37°C - Preperation of agarose gel (1%) analogous to 5.1.(see p.6) - 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 DNA-1KB-MARKER \\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested - Gelelectrophoresis at 120 V with regular controls - DNA-fragments of unknown origin were found 4. Overnight Culture of Strains 4196 and K699 for transformation - 3 ml YEPD Medium was given to each of 4 reaction tubes - Two yeast colonie 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 DNA of preparation 3.1 for transformation created Reagent Volume for one sample Eco RV 1 µl Buffer R 5 µl Add 20µl H2O 34 µl Σ 40 µl - Incubation over night at 37°C 6. Overnight culture of YIplac204 - 5 ml of Ampicilin-media (80µg/ml) wer inoculated with one colony of 1.2 pg 2 - Incubation at 37°C overnight Day 7, 03.07.15 1. Transformation with KCPlac22 and KlPlac202 ????? - over night culture 1:100 with MQ diluted with 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 under flame protection A: 50 ml B: 25 Einheit? → Media wrong estimated - 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 under shaking 2. Miniprep with KCPlac204 - over night culture (previous day) (6/6) in 2 ml Eppi transferred - centrifugation (700 rpm, 5 min) - 3 times repeated - pellet in 90 ml GTE+RNAv transferred - vortex - 100 µl NaOH/SDS Sx inverted - 135 ml KOAC/HoAc transferred Several times inverted - 80 µl NaHO/SDS added - centrifugation (15 min, 14000 rpm, 8 °C) - supernatant in tube transferred - in 1 ml 100 % EtOH transferred - centrifugation (10 min, 13300 rpm) - 1 ml 70 % ethanol added and removed - step repeated - ethanol dripped down and ?????? at room temperature 3. Gelelectrophoresis - 1,2 % agarose gel from the rest ? - from over night digestion (6/5) 5 µl given on gel - ????????????????? - after 1 h at 120 V gel picture - Midiprep from 7/2 used - Scheme marker // Midiprep - after ca. 2 h at 120 V gel picture - ????????????????? - Amount of proteins …. 4. Transfrmation of the yeast …... G99 - 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) - pellet in 25 ml …..... - pelletised at 3500 rpm and room temperature - pellet in 1 ml of 100 mM LiAc resuspended and given in Eppis - centrifugation (10 s, 3500 rpm, room temperature) - pellet in xxxx 100 mM LiA resuspended on volume of 500 ml - for each transformation 50 µl cell suspension (4) in ?????? - centrifugated for 10 s and supernatant was discarded/ as well as all probes of 4196 (too less xxxx) - mix of transformation added → 240 µl 50 % PEG 3350 → 36 µl 1 M LiAc → 5 µl carrierDNA (10mg/ml) → 4 µl YEP122 (ex) 5 µl YIP203?????????????? (negative control) → ??? µl of water to YEP122 → 360 µl ?????????????????????? → 360 ml 66 µl ddH2O to YCP22 → 360 µl 64 µl ddH2O to negative control → 360 µl - probe vortexed/ pellets diluted - incubation at room temperature (30 °C) - heat shock for 20 min at 42 °C - centrifugated for 10 s, pellet resuspended in 400 µl H2O → YEP122 200 µl plated → YEP204 400 µl plated → YEP22 200 µl plated → negative control 200 µl plated - incubated (72 h, 30 °C) 5. Precipitation of plasmid-DNA ???????????? - to DNA solution xxxx µl NaAC added - xxx µl ???? added - centrifugation (fullspeed, 5 min, room temperature) - DNA appears as a small band - remove supernatant - pellet washed in two volumes (240 µl) EtOH 70 % - dried at room temperature for 30 min - DNA solved in 5 µl TE - for experiment 7/4 used Day 8, 06.07.15 1. Examination V7/4 - colonies grown! - concept works 2. Resuspension of IDT geneBricks xxxxxxxxxxxxx and xxxxxxxxxx - 500 ng in 50 µl TE given (c = 10 ng/ml) - incubated (50 °C, 20 min) - vortexed and centrifugated (10 s to fullspeed) 3. Restriction diggestion for ligation of xxxxxxxxxxxx and xxxxxxxxx - needed amount of DNA → ??????????????? → ??????????????? → ??????????????? → ??????????????? - following restriction probes were constructed: insert his_spacer_adh1/ insert His_Rep_kl DNA 40 µl MM for 3 EcoRI 2 µl 6 µl PstI 2 µl 6 µl Buffer0 20 µl 60 µl 200 ddH2O 136 µl 408 µl Vector YEOlac181/ YCPlac22/ YEPlac204C7/3 DNA 5 µl MM for 4 RNAse 1 µl 4 µl EcoRI 1 µl 4 µl PstI 1 µl 4 µl Buffer0 5 µl 20 µl 50 addH2O 37 µl 148 µl Vector K801000 DNA 40 µl RNAse 1 µl EcoRI 2 µl PstI 2 µl Buffer0 10 µl 100 add H2O 45 µl - large probes were chosen because of the high ?????-concentration - over night incubation at 37 °C 4. Speedjet PCR-cloning with cccccccccc and xxxxxxxxxxx - as a backup the following speedjet probes were generated → 2 µl 10 x ligase buffer → 2,3 µl DNA solution → 1 µl vector → add 19 µl ddH2O → 13,5 → 1 µl TKLigase → ?????? → incubation (ca. 10 min) 5. Transformation - Dox-E.colis unfreezed - each 5 µl from 8/4 given on 50 ml competent cells (as positive control ???? on E.coli, as negative control no changes done) - ca 15 min given 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, 07.07.15 1. Analysis of over night ?????? from 8/3 - 1 % agarose gel ??????? (1 g 100 ml THE, 8 µl ???) - each 10 % put on gel with ????? buffer, that means YEP181/ YCPlac22/ YIlac204 → 5 µl K801000 → 10 µl inserts → 20 µl - each with 6x ????? buffer xxxxx 2. Restriction digestion 204 - 40 µl of plasmid solution 204 EcoRI digested sample from 7/2 S:12 → DNA 40 µl EcoRI 2 µl ???? 2 µl Buffer0 20 µl H2O 136 µl → 200 µl - incubation (2h, 37 °C) 3. Gels for extraction - 1 % gel 100 nl, middle chamber, large comb filled with total digest from 8/3 8µl Roth - 1 % gel 70 ml, small chamber, first comb ???????????? 4. Gelextraction - middle gel loaded with 45 ml VectorDNA - xxxxxxxxxxxxx - electrophoresis for 2 h at 50 V - ????????? - gel fragment weighted: xxxx mg - 3 time higher volume of QC-buffer added → 1+10 µl QC-buffer added - 10 min at 50 °C gel xxxx - after 3 min and 7 min for 5-7 s vortexed - 450 µl isopropanol added - probe inverted and shortly vortexed - 800 µl on speedcolumn on ?????????? - centrifugation (13300 rpm, 1 min) → flow rate discarded - step 3 times repeated - 0,75 ml PE buffer given on column for cleaning - centrifugation (13300 rpm, 1 min) - flow rate discarded - centrifugation (13300 rpm, 1 min) - flow rate discarded - column transferred on 1,5 ml tube - 30 µl ??????????? given on column - centrifugation (13300 rpm, 1 min) - eluate used for further experiments 5. Insertcleaning with PcR-Purification-Kit - on 180 µl 900 µl PB buffer given - 800 µl probe given on column - column centrifugated (13300 rpm, 1 min) in ?????????????? - remaining xxxx µl given on column - column centrifugated at 13300 rpm - both times flow rate was discarded - column loaded with 750 µl PE - centrifugation (13300 rpm, 1 min) - flow rate discarded - centrifugation (13300 rpm, 1 min) - flow rate discarded - column transferred on 1,5 ml Eppi - 30 µl BE (elution buffer) given - centrifugation (13300 rpm, 1 min) - eluate used for further experiments 6. Controlgel of eluate and digestion of 204 9/2 - filled on samll from 9/3 - ?????: Standard // Vector 22 // Digestion 204 // empty // Standard // Insert of Reporter 6 µl 3 µl 20 µl 6 µl 2,8 µl - gelelectrophoresis for 45 min - note: insert was added after 20 min gel current time 7. ÜNR - 2 colonies on plate 204, Pjet_His_spacer, Pjet_reporter_klein picked - to each 5 ml 80 µg/µl ampiciline medium given - over night incubation at 37 °C Day 10, 08.07.15 1. Ligation of the linearised vector VCPlac22 xxxxxxxxxxxxxxx - 3 probes for diligation generated → ligation with insert - 3 µl vector (YCPlac22) linearized - 14 µl insert (His3 spacer) linearized - 2 µl ligase buffer - 1 µl T4 Ligase → Re-Ligation control without insert - 3 µl vector (YCPlac22) linearized - 14 µl add H2O - 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 for 3 h at room temperature incubated 2. Miniprep from (9/7 S 20) - 2 ml from over night culture in 2 ml-Eppi transferred - centrifugation (7000 rpm, 5 min), supernatant ???? - 2 ml from over night culture in appropriate Eppis from step 1 transferrred - centrifugation (7000 rpm, 5 min), übereinander, supernatant ???? - pellet in 250 µl xxx resuspended - 250 µl buffer P2 added - 5 times inverted - 350 µl buffer N3 added - 5 times inverted - cemtrifugation (13000 rpm, 10 min) - supernatant loaded on column - centrifugation (13000 rpm, 1 min) - flow rate discarded - 750 µl PE buffer added - centrifugation (13000 rpm, 1 min) - flow rate discarded - centrifugation (13000 rpm, 1 min) - flow rate discarded - columns loaded in new Eppis - 50 µl elution buffer added - 1 min resting - centrifugation (13000 rpm, 1 min) 3. Digestion of Miniprep - ??????????? → 1 µl DNA xxxxxxxxxxxxxx → MM for 7 probes for each 19 µl 0,5 µl EcoRI 3,5 µl EcoRI 0,5 µl PstI 3,3 µl PstI 2 µl Buffer0 14 µl Buffer0 16 µl ddH2O 112 µl ddH2O - digestion for 3 h at 37 °C 7. Gelelectrophoresis of the Digestion from 3 - 20 µl from digestion taken and with xxxxxxxxxxxxxx - gelelectrophoresis (1 h, 120 V) - ??????????? ??????????? ?????????ß? Day 11, 09.07.15 1. Culture picking for over night culture - 12 colonies from 100 µl xxxxx taken (compare S2310/6) with pipette peak - in 2 ml 80 µg/ml ampiciline medium given - 2 colonies picked from YFP-plate - 5 ml 25 µg/ml Cm medium inoculated - over night incubation at 37 °C Day 12, 10.07.15 1. Miniprep of the over night culture from 11/1 - over night culture transferred in 2 ml Eppi - centrifugation (7000 rpm, 5 min) - ???????????????? - supernatant discarded - performed like 3/1 S 3 - ???????????? 2. Restriction digestion - ??????????? for all Minipreps (14) → 3 µl DNA-solution MM for 15 0,5 µl EcoRI 7,5 µl EcoRI 0,5 µl PstI 7,5 µl PstI 2 µl Buffer0 30 µl Buffer0 14 µl ddH2O 210 µl ddH2O - incubation (1,5 h, 37 °C) 3. Gelelectrophoresis - 2 gels (one with 100 ml and one with 70 ml) ???? - 13,6 µl Roth Ethidiumbromide added - probes from 12/2 with 4 µl ??????????????????????? - ????? as followed: → Gel I: 5 x empty // standard // YCPlac22 + insert 1 // 2 // 3 // 4 // 5 // 6 // 7 // 8 // → Gel II: standard // YCPlac22 + insert 9 // 10 // 11 // 12 // empty // YFP 1 // YFP 2 // stamdard // - gelelectrophoresis for 1 h - over night culture ???????????????? Day 13, 13.07.15 1. Miniprep via Quiagen column - 5 ml over night culture from 12/3 centrifuged (7000 rpm, 5min) in 2 ml Eppis ???????? - Quiagen Miniprep analogue to 10/2 performed (see page 21) 2. Restriction digestion - ???? probes from 13/1 (see above) EcoRI PstI digested Satz komisch - MM created → single Mastermix for 13 probes 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) 3. Gelelectrophoresis - one 1 % agarose gel 100 ml moulded - after cooling 8 µl ethidium bromide added - 14 ??????? used - to 12 probes from 13/2 each 4 µl ????????? added - 24 µl loaded on gel according to following scheme standard // YCPlac 22 + insert 1 // YCPlac 22 + insert 2 // YCPlac 22 + insert 3 // YCPlac 22 + insert 4 // ... YCPlac 22 + insert 12 // 4814 for IS - gelelectrophoresis (1 h, 120 V) Day 14, 14.07.15 1. Restriction digestion XhoI, Sal I from YCPlac22 + insert10 (13/13) - restriction digestion conducted → 20 µl DNA YCPlac22 + insert10 from day 13 2 µl Sal I 4 µl XhoI 10 µl Buffer0 64 µl ddH2O - incubation (2 h, 37 °C) 2. Addition of restriction sites via PCR probe water control H2O 71 µl 73 µl 5 x buffer 20 µl 20 µl template - 2 µl ???? 2 µl 2 µl revPrimer 2 µl 2 µl ???? 1 µl 1 µl Σ 100 µl Σ 100 µl → next time less - 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 3. Gelelectrophoresis of the restriction digestion and the PCR - 70 ml 1 % agarose gel moulded - after cooling 8 µl ethidium bromide added - from PCR 5 µl with 1 µl ?????????????? - to digestion 4 µl ????? added - according to following scheme loaded → standard – digestion – water control – PCR - gelelectrophoresis (1 h, 120 V) 4. Purification of the PCR fragment - analogue to 9/5 page 19 - pellet was solved in 30 µl EB 5. Restriction digestion ofthe YFP-PCR fragment - purified insert digested with XhoI Sal I → 30 µl DNA 2 µl Sal I 4 µl XhoI 10 µl Buffer0 54 µl ddH2O - incubation (3 h, 37 °C)) 6. Gelextraction of the digested vector - gelelectrophoresis (1,5 h) in 1 % agarose gel analogue 9/4 page 19 - band with scalpel removed and weighted = 390 mg - three fold amount of QC added (1160 µl) - analogue to 9/4 page 19 conducted Day 15, 15.07.15 1. Purification of the digestion from 14/5 - analogue to 9/5 page 19 - 380 µl PB used - in 30 µl solved da fehlt iwas 2. Dephosphorylation of the vector - alkaline phosphatase mix created → 20 µl TCPlac22 + insert XhoI Sal I digested iwas fehlt 3 µl FAP buffer 1 µl FAP 6 µl H2O - incubation (20 min, 37 °C) - incubation (5 min, 75 °C) 3. Ligation YCPlac22 + insert ????? and YFP - 3 liagtionansätze prepared A) ??? µl vector, dephosphorylated (YCPlac22) 5 µl insert (YFP) 2 µl ligase buffer 1 µl T4 ligase 8 µl ddH2O B) ??? 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) 4. Transformation of the ligation - analogue to 8/5 conducted - transformation as followed plated 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 ??? and H2O on ampiciline plated Day 16, 16.07.15 1. ?????? - Bleistift - 20 colonies of the colonies picked and in each 5 m 100 µg/ml ampiciline medium given da passt iwas nicht → over night culture at 37 °C 2. Trafo from 15/3 repeated - anologue to 15/3 conducted Day 17, 17.07.15 1. Miniprep of the over night culture - anologue to 3/1 conducted 2. Restriction digestion from the Miniprep - restriction ??????? created → 1 µl Miniprep MM for 21 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 - digestion (2 h, ??? °C) 3. Gelelectrophoresis - each probe with 4 µl ?????? - ??? µl ???? - 6 µl standard loaded - scheme: standard // Miniprep 1 – 10 = Gel I standard // Miniprep 11 – 20 = Gel II - expected fragments ~ 5000 bp ~ 700 bp