Yeast transformation with YFP

Day 1, 15/06/25:

1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of hostlab's strain collection

• Strains taken from -80°C freezer were spread out on YPED-Medium plates
• Incubation for 3 days at 26°C

2. Transformation of Vectors YCplac22, YEplac 181 and YIplac204 in 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, 15/06/26:

1. Picking colonies for overnight cultures (ONK)

• Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000)
• Afterwards incubation overnight at 37°C

Day 3, 15/06/29:

1. DNA-preparation via alkaline Lysis

• Experimental procedure according to " Protocol 1: Alkaline Lysis "
• Changes to protocol:
  → No centrifugation of ONK
  → Two 2ml reaction tubes filled with ONK instead

2. Picking of yeast colonies

• Two yeast colonies picked out of YPED
• Medium plates from Day 1 (see day 1/1.)
• Clone 1 named A
• Clone 2 named B

Day 4, 15/06/30:

1. Restriction digest of the obtained DNA-Solutions

• Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table

• 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:

• Digests were incubated at 37°C for 3 h

2. Gelelectrophoresis of digested DNA

• Gelectrophoresis was executed according to "protocol 2: Gelelectrophoresis "
• 2 µl 6x staining solution were given unto 10 µl digest
• Samples were loaded onto the gel according to following scheme
  ⇒ 1kb-DNA-Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\DNA
• 1kb
• Marker (6µl)
• Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes <\ul> hier Bilder 150630a
• c einfügen Figure 1: Gelphoto taken 1 hour after start.. Estimated fragment sizes were for YCpla22 (total: 4854) = 2171bp and 2683bp, YEplac181(total: 5741 bp)= 3170bp and 2571bp, YIplac204 (total:3545)= 2381 bp and 880 bp as well as Bba_K801000 (total: 4923 bp)= 3043bp, 1390 and 490bp. All except YIplac204 and Bba_K801000 show estimated dna bands. Picture was digitally altered by removing stains in the left corner and including RNA
• Description as well as cropping. Figure 2: Gelphoto after 1h 40 minutes. Figure 3: Gelphoto after 2h 20 minutes.

  Day 5, 15/07/01:

  1. Repetition of restriction digest for Ycplac 204 and K801000 digested

  • Mastermix created for BBa_K801000 and YIplac204

    Reagent	Volume for one sample<\th>	Mastermix for three samples
    Eco RI	0.5 µl	1.5 µl
    EcoRV	0.5 µl	1.5 µl
    Tango Buffer	4 µl	12 µl
    Add 20µl ddH2O	13 µl	39 µl
    Σ	18 µl	54 µl

  • 2µl of obtained DNA
  • Solution were transfered to 18 µl of mastermix
  • Incubation at 37°C for 3 hours
  • Gelelectrophoresis was conducted according to protocol 2
  • 2 µl of 6x staining solution were added onto 10 µl of digest
  • Samples were loaded in pockets according to following scheme ⇒ 6µl 1kb-DNA-marker// YIplac204 digested (15/07/01) // K801000 digested(15/07/01)//YCplac22 (15/06/30)// K801000(15/06/30)// YIplac204 (15/06/30)//empty//YEplac181 (15/06/30)// empty // 6µl 1kb-DNA-marker Bild 150701a aus Figure 4: Photo of gelelectrophoresis at 120 V after 50 minutes.

  2. Preparation of S. Cerevisiae tryptophan negative plates

  • Added following substances in two 1 liter flasks:
    • 3.35g Difco yeast nitrogen base 2/o aminoacid
    • 5.5 g CAA vitamin assay
    • 10 g Glucose
    • 83.0 mg Tyrosin
    • Uracil
    • Adenin
    • mix
    • 50.5 mg Leucin
    • 22g Agar

  3. Preparation of S. Cerevisiae full media plates

  • Added following substances in two 1 liter flasks
    • 5.3g yeast extract
    • 11g Bacopepton
    • 10g Glucose
    • 22.7 mg Adenin
    • 11g Agar

  4. Overnightculture of YIplac204 positive bacteria

  • two times 5 ml ampicilin medium (80µg/ml)inoculated with 2 ampicilline ressistant bacteria colonies picked of plate from day 1/2.
  • Incubation at 37°C over night

  Day 6, 15/07/02:

  1. Moulding of Agar-plates

  • Added 500ml destilled Water to each Flask of day 5/2. and day 5/3.
  • Short mixing with stirring bar
  • Flask autoclaved
  • Stirring with stirring bar for 20 minutes at room temperature
  • Moulding of plates underneath a laminar flow hood

  2. DNA-Preperation of overnight culture (see day 5/4.)

  • Conducted analogous to day 3/1. according to protocol 1

  3. Digest of obtained DNA

  • Mastermix created to digest 2 µl DNA solution
    

    Reagent	Volume for one sample	Mastermix for three samples
    EcoRV	0.5 µl	1.5µl
    Buffer R	2.0 µl	6 µl
    Add 20µl H2O	15.5µl	46.5µl
    Σ	18µl	18µl

    
    
  • 2 µl DNA DNA
  • preperation of each dublicat of YIp204 were added onto 18µl mastermix
  • Incubated for 3 hours at 37°C
  • Electrophoresis was conducted according to protocol 2
  • Added 4µl 6x staining buffer to each digest after incubation time
  • Added 4µl 6x staining buffer to 20µl undigest DNA
  • Preperation
  • Loaded gel according to following scheme
    &rArr 6µl 1kb-DNA-marker\\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested\\
  • DNA-fragments of unknown origin were found Bilder unter 150702a http://www.studon.uni
  • erlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode=

  4. Overnight culture of strains 4196 and K699 for transformation

  • 3 ml YEPD Medium was given to each of 4 reaction tubes
  • Two yeast colonies of 4196 and K669 were picked
  • Incubation at 30°C over night

  5. Restriction digest of Vector DNA for the transformation

• Mix to digest 10 µl YIplac204 DNA obtained in preparation 3.1 for transformation created

  Reagent	Volume for one sample
  Eco RV	1 µl
  Buffer R	5 µl
  Add 20µl ddH2O	34 µl
  Σ	40 µl

• Incubation over night at 37°C

6. Overnight culture of YIplac204

• 5 ml of ampiciline
• media (80µg/ml) were inoculated with one colony obtained of experiment Day1 2. (p.: 2)
• Incubation at 37°C overnight

Day 7, 15/07/03

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

2. DNA

Preperation with YIplac204 overnight culture (see day 6/6.)

• Experiment conducted according to protocol 1

3. Gelelectrophoresis of overnight digestion and DNA Preparation

• Experiment conducted according to protocol 2
• hanges 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 Hier 150703a einfügen ort http://www.studon.uni
• 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.
• After the photo was taken
• DNA
• Preperation from day 7/2. loaded on same gel
• Scheme : 6 µl 1kb-DNA-marker //overnight digest //empty// marker // Miniprep Hier 150703b einfügen ort: http://www.studon.uni
• 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.

4. Precipitation of plasmid-DNA of digested YIplac204

• 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

5. Transformation of the yeast K699

• 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)

Day 8, 15/07/06:

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:

  1. 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. Speedjet PCR

cloning with his3_rep_klein and his_spacer_adh1

• as a backup the following speedjet Samples were generated

• Incubation (ca. 10 min)

5. Transformation

• DH5alpha
• E.colis 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 <\h2>

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)