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 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
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 |
&Sigma | | 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 (see: p. 2) were given in one mastermix tube each
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
Digests were incubated at 37°C for 3 h
\subtitle 2. Gelelectrophoresis of digested DNA
Gelectrophoresis was executed according to protocol 2
2 µl 6x staining solution were given unto 10 µl digest
Samples were loaded onto the gel according to following scheme
=> DNA1kbMarker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\DNA1kbMarker (6µl)
Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes
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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 RNADescription 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
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
2µl of obtained DNASolution 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 DNA1KBMARKER// 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 DNA1KBMARKER
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Figure 4: Photo of gelelectrophoresis at 120 V after 50 minutes.
\subtitle 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 TyrosinUracilAdeninmix
50.5 mg Leucin
22g Agar
\subtitle 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
\subtitle 4. Overnightculture of YIplac204 positive bacteria
two times 5 ml ampicilin medium (80µg/ml) with 2 ampressistant bacteria picked of plate from 1.2 inoculated
Incubation at 37°C over night
Day 6, 15/07/02:
\subtitle 1. Moulding of Agarplates
Added 500ml destilled Water to each Flask of 5.2 and 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
\subtitle 2. DNAPreperation of overnight culture (see 5.4. p.:)
Conducted analogous to Day 3.1. according to protocol 1
\subtitle 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 6µl
Add 20µl H2O 15.5µl 46.5µl
Sum 18 µl 18 µl
2 µl DNA DNApreperation 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 DNA1KBMARKER \\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested
DNAfragments of unknown origin were found
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\subtitle 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
\subtitle 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
Sum 40 µl
Incubation over night at 37°C
\subtitle6. Overnight culture of YIplac204
5 ml of ampicilinemedia (80µg/ml) were inoculated with one colony obtained of experiment Day1 2. (p.: 2)
Incubation at 37°C overnight
Day 7, 15/07/03
\subtitle 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
\subtitle 2. DNAPreperation with YIplac204 overnight culture (see 6.6)
Experiment conducted according to protocol 1
\subtitle 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 1kbDNAmarker \\ overnight digest
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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
DNAPreperation from Day 7 2. ( see page 10) loaded on same gel
Scheme : 6 µl 1kbDNAmarker //overnight digest //empty// marker // Miniprep
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Figure: Gelelectrophoresis of preparated vector YIplac204 (3545 bp). No contamination detected. DNA concentration estimated to be over 70 ng/µl.
\subtitle 5. Precipitation of plasmidDNA 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
\subtitle 4. 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:
\subtitle 1. Examination of 7 4.
Colonies grown!
Transformation works
\subtitle 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)
\subtitle 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= 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
large volumes were chosen because of the high EDTAconcentration
over night incubation at 37 °C
4. Speedjet PCRcloning with his3_rep_klein and his_spacer_adh1
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 > 13.5µl
1 µl T4Ligase
Incubation (ca. 10 min)
5. Transformation
DH5alphaE.colis unfreezed
Each 5 µl from Day8 4. given on top of 50 µl competent cells
As a positive control 1µl PBSCBluescript 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
\subtitle 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 )
K801000 > 10 µl (2 µl )
Inserts(his3_rep_klein and his_spacer_adh1) > 20 µl (4 µl )
Pockets were loaded according to following scheme
=> 1 kb DNAmarker //YEplac181//YCplac22//YIplac204//K801000//his_spacer_adh1//his3_rep_klein
hier Bild 150707a einfügen Ordner
figure: Linearisation of all vectors achieved. DNAconcentration of Reporter Insert his3_rep_klein below detection limit. Insert his_spacer_adh1 only just above the detection range.
\subtitle 2. Restriction digestion 204
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
incubation (2h, 37 °C)
\subtitle 3. Gelelectrophoresis for extraction
Gelelectrophoresis conducted according to protocol 2
Middlepocket of gel loaded with 45 µl YCplac22 Day 8 3.according to following scheme:
6µl 1 kb DNAmarker // empty // YCplac22 // empty
Changes to protocol:
> large comb used
> run for 2h at 50V
\subsection 4. Gelextraction
Gel fragment weighted: 470 mg
3 times the volume QCbuffer added > 1410 µl QCbuffer added
10 min at 50 °C gel dissolved
After 3 min and 7 min for 57 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
\subsection 5. Further cleaning with PCRPurificationKit
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
\subsection 6. Controlgelelectrophoresis of eluate and digestion of YIplac204 and YCplac22 Day 9 2.
Gelelectrophoresis according to protocol 2
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
Changes to protocol:
> Gelelectrophoresis for 45 min
> note: insert was added after 20 min
Hier Bild 150707b aus dem Gelphoto ordner
Figure: Controlgel after purification. Vector DNAconcentration is still above the 25ng per µl. Detection of the Insert his_rep_klein was possible.
\subsection 7. Overnight culture of Pjettransformed E. coli
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:
\subsection 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
\subsection 2. Miniprep from overnight culture Day 9 7.
2 ml over night culture transferred to 2 mlreaction 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
DNAPreparation conducted as given by QuiaGen Miniprep instruction
Elution in 50 µl elution buffer
\subsection 3. Digestion of Miniprep
Digest prepared
> 1 µl DNA from the miniprep (see p.:19:) > 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
\subsection 4. Mouding of Chloramphenicol agar plates
TY Medium with agar heated
Cooling while mixing
Addition of 50 µl Chloramphenicol (100 mg/ml)> c_end= 10µg/ml
Moulding of plates
\subsection 5 Preparation of Xgal plates
40µl 2% xgal spread on 6 ampicilin enriched plates
40µl IPTG spread on the dried plates
40µl dH2O spread on the dried plates
\subsection 6 Transformation of Ligation and EYFP (BBa_E2030)
Each of folowing DNAsamples added to 50 µl DH5alpha 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 \textit{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
\subsection 7. Gelelectrophoresis of the Digestion from 3. (see above)
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:
his3_rep_klein A // his3_rep_klein B // his_spacer_adh1 A // his_spacer_adh1 B// YIplac204 A //YIplac 204 B// 1kb DNAmarker
Foto: 150708a.png
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Bildunterschrift. Gelfoto of pjet digest and control of YIP204 digest.
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 YFPplate
5 ml 25 µg/ml Canamycin medium inoculated
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)
conducted according to protocol 1
eluted in 30µl 1x TE
\subsection 2. Restriction digestion
Restriction for all DNAPreparation (14)
> 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
incubation (1.5 h, 37 °C)
\subsection 3. Gelelectrophoresis
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 DNAmarker // YCPlac22 + insert 1 //
2 //
3 //
4 //
5 //
6 //
7 //
8 //
> Gel II: 1kb DNAmarker // 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.
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
5 ml over night culture from Day 12 3. centrifuged (7000 rpm, 5min) in 2 ml Eppis
Quiagen Miniprep analogous to 10 2. performed
\subsection 2. Restriction digestion
DNASolutions 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)
\subsection 3. Gelelectrophoresis
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 DNAmarker //
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)
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)
\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
Forward Primer 2 µl 2 µl
Reverse Primer 2 µl 2 µl
Fusion Polymerase 1 µl 1 µl
Sum 100 µl 100 µl
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
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 PCRsample
2 µl staining solution added to 10 µl of digest
According to following scheme loaded
1kb DNAmarker // 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
Analogous to Day 9 5. (p.:16)
pellet was solved in 30 µl EB
\subsection 5. Restriction digestion of the YFPPCR 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))
\subsection 6. Gelextraction of the digested vector
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
\subsection 1. Purification of the digestion from 14/5
Analogue to Day 9 5.
Changes to Day 9 5.
> 380 µl PB used
> eluted in 30 µl Elution Buffer
\subsection 2. Dephosphorylation of the vector
alkaline phosphatase mix created
> 20 µl TCPlac22 + insert XhoI Sal I digested
3 µl FAP buffer
1 µl FAP
6 µl H2O
incubation (20 min, 37 °C)
incubation (5 min, 75 °C)
\subsection 3. Ligation YCPlac22 + insert his3_rep_klein and YFP
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)
\subsection 4. Transformation of the ligation
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 PBSCBluescript and H2O on ampiciline plated
Day 16, 15/07/16
\subsection 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
\subsection 2. Retransformation of the ligation
Repition of Day 15 3. (see above)
Day 17, 15/07/17
\subsection 1. Miniprep of the over night culture
Performed according to protocol 1
\subsection2. Restriction digestion from the Miniprep
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
digestion (2 h, 37 °C)
\subsection 3. Gelelectrophoresis
Gelelectrophoresis performed according to protocol 2
4 µl staining solution was added to each digest
6 µl 1kb DNAmarker loaded
scheme: 1kb DNAmarker // Miniprep 1 10 = Gel I
1kb DNAmarker // Miniprep 11 20 = Gel II
expected fragments ~ 5000 bp
~ 700 bp
Bilder einfügen 150717a und 150717b
Beschriftung
a)GelI. Restriction sites XhoI and SalI are compatible, but after XhoISalIligation the restriction site is removed. Thus "homo"ligation creates an insert after XhoISalI digestion whereas "hetero"ligation does not.
b)GelII Restriction sites XhoI and SalI are compatible, but after XhoISalIligation the restriction site is removed. Thus "homo"ligation creates an insert after XhoISalI 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 YEPDMedium each
Incubation overnight at 28°C
Day 19, 15/07/21
1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22YFP
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
7196 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)/ YCplac22YFP 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 Adrian
1 von Filip
1 von Vlady (schon auf Studon)
1 von Frederike