Team:FAU Erlangen/Tour32
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: