Team:FAU Erlangen/Tour32
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 day 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 expression cassette (EC) and target cassette (TC) (TC)
- 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 expression cassette (EC) and target cassette (TC) (TC)
- 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 TC for cloning in YIplac204 =200 ng (plasmid) * 700 bp/3500 bp *5 = 200ng Insert TC for cloning in YCplac22 =200 ng (plasmid) * 700 bp/4850 bp *5 = 144 ng Insert EC for cloning in YEplac181 =200 ng (plasmid) * 500 bp/5740 bp *5 = 86ng Insert EC for cloning in K801000 =200 ng (plasmid) * 500 bp/5500 bp *5 = 90ng
- following restriction digests were constructed:
- insert EC/ insert TC
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 target cassette (TC) and EC
- 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 (target cassette (TC)/ EC
20 µl
(4 µl)
- Pockets were loaded according to following scheme
⇒ 1 kb DNA-marker //YEplac181//YCplac22//YIplac204//K801000//EC//target cassette (TC)
- 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 target cassette (TC)
- 3 samples for ligation generated
- ligation with insert
- 3 µl vector (YCPlac22) linearized
- 14 µl insert (target cassette (TC)/ EC) 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
Filip Teil 1blub
Generation of TALE and beginning of generating RPD3 constructsblub
Generation of RPD3 constructsblub
Results of the project2015-09-10
- Viewed ONCs with fluorescence microscope: 699 (negative control) shows strong fluorescence. Probably contaminated with YFP yeast. Plated on –Trp plate to test the assumption.
- Picked 699 clone from fresh plate (M. Dahl) – clone shows no fluorescence.
- Transferred ONCs to 50ml of the appropriate deficient SC medium. Also inoculated one clone from 699 plate (M. Dahl) in 50ml SC full medium. Additionally inoculated 699 strain in –Trp SC and –Ura –Leu –Trp SC to see if it survives. Incubation over night at 28°C and 180rpm
2015-09-11
- RNA extraction from yeast overnight cultures using the RiboPure Yeast RNA isolation Kit. Handling according to manufacturer’s instructions. Executed by F. Winter and N. Übelmesser.
- A sample was taken from 699 and YIYFP1 isolates before DNase treatment to test via PCR for YFP DNA.
- All samples were tested with YFP PCR for residual gDNA. PCR over night.
2015-09-14
- PCR samples were loaded on a gel. Bands appeared in all samples except the 699 sample.
- Yeast plates from 09-09: Colonies on TALE1/YEplac181 plate, otherwise none.
- Mixed RT reaction according to protocol.
- 50µl reactions for +RT and 25µl reactions for –RT control.
2015-09-15
- Inoculated one colony of YIYFP1, 3 Rpd3/TALE constructs and 699 in SC medium at 30°C for 4 hours. Then the cultures were inspected via CLSM. All cultures show strong fluorescence. Most likely the SC medium is the cause.
- Tested qPCR primers: eYFP primer efficiency: 95%, tub Primer efficiency: 85%
2015-09-16
- Mixed qPCR Master mix (Promega goTaq qPCR Master Mix) for tomorrow. Test PCR of all RT reactions for YFP DNA. Bands in every sample except 699.
2015-09-17
- Pipetted all qPCR reactions on 96 well plate. qPCR reaction was successful.
- Inoculated yeast ONCs for CLSM tomorrow.
2015-09-18
- Data analysis of qPCR with J. Schmidpeter. Rpd3/TALE transformed yeast strains exhibit strongly lowered levels of YFP mRNA.
- One more qPCR to confirm primer efficiency. The result does not differ much from the last time.
- CLSM: Transformed YIYFP strains appear to show less fluorescence.
- following restriction digests were constructed: