Difference between revisions of "Team:FAU Erlangen/Tour32"
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Adrian FAU (Talk | contribs) |
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<li> Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table | <li> Mastermix for a control digest of the obtained DNA-Solutions of plasmids YCplac22, YEplac181, YIplac204 produced according to following table | ||
</ul> | </ul> | ||
| + | <br> | ||
<table> | <table> | ||
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<li> centrifugation (13300 rpm, 1 min) | <li> centrifugation (13300 rpm, 1 min) | ||
<li> flowthrough discarded | <li> flowthrough discarded | ||
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Revision as of 14:32, 18 September 2015
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
| 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
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
- Mastermix created for BBa_K801000 and YIplac204
- 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
- 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:
- insert his_spacer_adh1/ insert His_Rep_klein
- 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
DNA 40µl MM for 3 samples EcoRI 2µl 6µl PstI 2µl 6µl Buffer 0 20µl 60µl add 200µl ddH2O 136 µl 408 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
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)
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)
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 hier Bild 150707a einfügen Ordner figure: Linearisation of all vectors achieved. DNA- concentration of Reporter Insert his3_rep_klein below detection limit. Insert his_spacer_adh1 only just above the detection range.
2. Restriction digestion 204
- 40 µl plasmid solution 204 obtained out of Day 7 2. digested
<\ul>
DNA 40 µl EcoRI 2 µl PstI 2 µl Buffer O 20 µl H2O 136 µl Σ 200 µl - incubation (2h, 37 °C)
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 DNA - marker // empty // YCplac22 // empty
- Changes to protocol:
- large comb used
- run for 2h at 50V
4. Gelextraction via Quiagen kit
- 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
5. Further cleaning with Quiagen PCR-purification-kit
- 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
- following restriction digests were constructed: