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| − | <h1>Labprotocol: S. cerevisiae GFP</h1> | + | <p>1 von Adrian</p> |
| − | | + | <p>1 von Filip</p> |
| − | Inhaltsverzeichnis
| + | <p>1 von Vlady (schon auf Studon)</p> |
| − | Day 1: 3
| + | <p>1 von Frederike</p> |
| − | 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of strain collection 3
| + | |
| − | 2. Transformation of Vectors YCplac22, Yeplac 181 and YIplac204 in DH5α E. coli 3
| + | |
| − | Day 2: 3
| + | |
| − | 1. Picking colonies for overnight cultures 3
| + | |
| − | Day 3: 3
| + | |
| − | 1. Preperation via alkaline Lysis 3
| + | |
| − | Day 4: 4
| + | |
| − | 1. Restriction digest of the obtained DNA-Solutions 4
| + | |
| − | 2. Gelelectrophoresis of digested DNA 5
| + | |
| − | Day 5, 01.07.15: 7
| + | |
| − | 1. Repetition of restriction digest for Ycplac 204 and K801000digested 7
| + | |
| − | 2. Preperation of S. Cerevisiae tryptophan negative plates 8
| + | |
| − | 3. Preparation of S. Cerevisiae full media plates 8
| + | |
| − | 4. Overnightculture of YIplac204 positive bacteria 9
| + | |
| − | Day 6, 02.07.15 9
| + | |
| − | 1. Moulding of Agar-plates 9
| + | |
| − | 2. DNA-Preperation of overnight culture (see 5.4. p.:7) 9
| + | |
| − | 3. Controldigestion of obtained DNA 9
| + | |
| − | 4. Overnight Culture of Strains 4196 and K699 for transformation 10
| + | |
| − | 5. Restriction digest of Vector DNA for the transformation 10
| + | |
| − | 6. Overnight culture of YIplac204 11
| + | |
| − | Day 7, 03.07.15 11
| + | |
| − | 1. Transformation with KCPlac22 and KlPlac202 ????? 11 | + | |
| − | 2. Miniprep with KCPlac204 11
| + | |
| − | 3. Gelelectrophoresis 12
| + | |
| − | 4. Transfrmation of the yeast …... G99 12
| + | |
| − | 5. Precipitation of plasmid-DNA ???????????? 13
| + | |
| − | Day 8, 06.07.15 13
| + | |
| − | 1. Examination V7/4 13
| + | |
| − | 2. Resuspension of IDT geneBricks xxxxxxxxxxxxx and xxxxxxxxxx 13
| + | |
| − | 3. Restriction diggestion for ligation of xxxxxxxxxxxx and xxxxxxxxx 14
| + | |
| − | 4. Speedjet PCR-cloning with cccccccccc and xxxxxxxxxxx 15
| + | |
| − | 5. Transformation 15
| + | |
| − | Day 9, 07.07.15 15
| + | |
| − | 1. Analysis of over night ?????? from 8/3 16
| + | |
| − | 2. Restriction digestion 204 16
| + | |
| − | 3. Gels for extraction 16
| + | |
| − | 4. Gelextraction 16
| + | |
| − | 5. Insertcleaning with PcR-Purification-Kit 17
| + | |
| − | 6. Controlgel of eluate and digestion of 204 9/2 17
| + | |
| − | 7. ÜNR 17
| + | |
| − | Day 10, 08.07.15 17
| + | |
| − | 1. Ligation of the linearised vector VCPlac22 xxxxxxxxxxxxxxx 17
| + | |
| − | 2. Miniprep from (9/7 S 20) 18
| + | |
| − | 3. Digestion of Miniprep 18
| + | |
| − | 7. Gelelectrophoresis of the Digestion from 3 19
| + | |
| − | Day 11, 09.07.15 19
| + | |
| − | 1. Culture picking for over night culture 19
| + | |
| − | Day 12, 10.07.15 19
| + | |
| − | 1. Miniprep of the over night culture from 11/1 19
| + | |
| − | 2. Restriction digestion 19
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| − | 3. Gelelectrophoresis 20
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| − | Day 1:
| + | |
| − | 1. Cultivating S. cerevisiae strain K699 and BY4742 derivative 4196 out of 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
| + | |
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| − | Day 2:
| + | |
| − | 1. Picking colonies for overnight cultures
| + | |
| − | - Picking a colony for each vector and additionally from the agarstamp ordered from the registry (K801000)
| + | |
| − | - Incubation in 5ml ampicilin enriched medium (LB with 80 µg/µl) for 2 days at 8°C
| + | |
| − | - Afterwards incubation overnight at 37°C
| + | |
| − | | + | |
| − | Day 3:
| + | |
| − | 1. Preperation via alkaline Lysis
| + | |
| − | - Vortexing ÜNK (see 2.1./ p.: 2)
| + | |
| − | - Transferring 2 ml out of ÜNK in two 2ml reaction tubes thus creating a dublicate
| + | |
| − | - Centrifugation at 7000 rpm for 5 minutes (Centrifuge used: Haereus pico17; producer Thermo Fischer Scientific)
| + | |
| − | - Removed supernatant
| + | |
| − | - Pellets resuspended in 90µl GTE-Buffer (50 mM Glucose, 10 mM EDTA, 25 mM Tris/HCl pH8,0)
| + | |
| − | - Vortexing for ca. 10 seconds
| + | |
| − | - Addition of 180µl NaOH/SDS (1% SDS, 0,2 M NaOH)
| + | |
| − | - Inverting tubes 4 times
| + | |
| − | - Addition of 135µl Kac/Hac (3M KAc/2M HAc)
| + | |
| − | - Inverting 10 times
| + | |
| − | - Incubation for 5 minutes on ice
| + | |
| − | - Centrifugation at 13,300 rpm for 15 minutes
| + | |
| − | - Each supernatant transferred in one 1,5 reaction tube
| + | |
| − | - Addition of 1 ml EtOH (100%)
| + | |
| − | - Vortexing for 10 seconds
| + | |
| − | - Incubation at 13,300 rpm for 10 minutes
| + | |
| − | - Removal of ethanol
| + | |
| − | - Addition of 1ml EtOH (70%)
| + | |
| − | - Centrifugation at 13,300 rpm for 10 seconds
| + | |
| − | - Repitition of the last two steps
| + | |
| − | - Evaporation of remaining EtOH at room temperature
| + | |
| − | - Resuspending pellet in 40µl TE-Puffer
| + | |
| − | - Combining both duplicats of one sample
| + | |
| − | Day 4:
| + | |
| − | 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 table
| + | |
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| − | 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 H2O
| + | |
| − | 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 3.1; p: 2 were given in one mastermix tube each
| + | |
| − | - 1µl K80100 solution obtained in 3.1; p.:2 was added to following reagents:
| + | |
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| − | Reagent
| + | |
| − | Volume for one sample
| + | |
| − | Pst I
| + | |
| − | 0,5 µl
| + | |
| − | Buffer O
| + | |
| − | 2 µl
| + | |
| − | Add 20µl H2O
| + | |
| − | 16,5 µl
| + | |
| − | Σ
| + | |
| − | 19 µl
| + | |
| − | | + | |
| − | - Digest were incubated at 37°C for 3 h
| + | |
| − | | + | |
| − | 2. Gelelectrophoresis of digested DNA
| + | |
| − | - 2 µl 6x staining solution were given unto 10 µl digest
| + | |
| − | - 9µl H20 were given unto 1 µl DNA solution obtained in 3.1; p.:2 each and 2 µl 6x staining solution were added
| + | |
| − | - 1% agarose-gel was prepared by adding 1g agarose to 100 ml 1xTAE and heating in microwave oven
| + | |
| − | - 8µl Ethidium Bromid were added after cooling of solution
| + | |
| − | - Samples were added onto the gel according to following scheme
| + | |
| − | => DNA-1kb-Marker (6µl)\\YCplac22 undigested\\ YCplac22 digested\\YIplac204 undigested\\YIplac204 digested\\ YEplac181 undigested\\ YEplac181 undigested\\ empty (pocket damaged)\\ YEplac181 digested\\K801000 undigested\\K801000digested\\empty\\DNA-1kb-Marker (6µl)
| + | |
| − | - Gelelectrophoresis was conducted at 120 V
| + | |
| − | - Photos were taken 1 hour, 1 hour 40 minutes and 2 hours 20 minutes
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| − | Day 5, 01.07.15:
| + | |
| − | 1. Repetition of restriction digest for Ycplac 204 and K801000digested
| + | |
| − | | + | |
| − | -Mastermix created for K801000 and YCplac204
| + | |
| − | | + | |
| − | Reagent
| + | |
| − | Volume for one sample
| + | |
| − | Mastermix for three samples
| + | |
| − | EcoRI
| + | |
| − | 0,5 µl
| + | |
| − | 1,5 µl
| + | |
| − | EcoRV
| + | |
| − | 0,5 µl
| + | |
| − | 1,5 µl
| + | |
| − | Tango Buffer
| + | |
| − | 4 µl
| + | |
| − | 12 µl
| + | |
| − | Add 20µl H2O
| + | |
| − | 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
| + | |
| − | - 100 ml 1% agarose gel were prepared in microwave oven
| + | |
| − | - After light cooling 8µl Ethidium Bromid were added
| + | |
| − | - 2 µl of 6x staining solution were added onto 10 µl of digest
| + | |
| − | - Samples were loaded in pockets according to following scheme
| + | |
| − | | + | |
| − | => 6µl DNA-1KB-MARKER\\ YIplac204 digested (01.07.15) \\ K801000 digested(01.07.15)\\ K801000(30.06.15)\\ YIplac204 (30.06.15)\\empty\\YEplac181 (30.06.15)\\empty\\ 6µl DNA-1KB-MARKER
| + | |
| − | - Gelelectrophoresis at 120V for 50 minuts
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| − | 2. Preperation 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
| + | |
| − | | + | |
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| − | 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) with 2 amp-ressistant bacteria picked of plate from 1.2 pg 2 inoculated
| + | |
| − | - Incubation at 37°C over night
| + | |
| − | | + | |
| − | Day 6, 02.07.15
| + | |
| − | 1. Moulding of Agar-plates
| + | |
| − | - Added 500ml destilled Water to each Flask of 5.2 and 5.3 p.:7
| + | |
| − | - 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 5.4. p.:7)
| + | |
| − | - Conducted analogous to 3.1. (see p.: 3) with a dublicat
| + | |
| − | | + | |
| − | 3. Controldigestion of obtained DNA
| + | |
| − | | + | |
| − | - Mastermix created to digest 2 µl DNA solution
| + | |
| − | | + | |
| − | Reagent
| + | |
| − | Volume for one sample
| + | |
| − | Mastermix for three samples
| + | |
| − | EcoRV
| + | |
| − | 0,5 µl
| + | |
| − | 1,5 µl
| + | |
| − | Buffer R
| + | |
| − | 2 µl
| + | |
| − | 6 µl
| + | |
| − | Add 20µl H2O
| + | |
| − | 16,5 µl
| + | |
| − | 46,5 µl
| + | |
| − | Σ
| + | |
| − | 18 µl
| + | |
| − | 54 µl
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| − | | + | |
| − | | + | |
| − | - 2 µl DNA DNA-preperation dublicat of YIp204 were added onto 18µl mastermix
| + | |
| − | - Incubated for 3 hours at 37°C
| + | |
| − | - Preperation of agarose gel (1%) analogous to 5.1.(see p.6)
| + | |
| − | - 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
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| − | => 6µl DNA-1KB-MARKER \\ Prep A digested\\ Prep B digested \\ Prep A undigested \\ Prep B undigested
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| − | - Gelelectrophoresis at 120 V with regular controls
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| − | - DNA-fragments of unknown origin were found
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| − | 4. Overnight Culture of Strains 4196 and K699 for transformation
| + | |
| − | | + | |
| − | - 3 ml YEPD Medium was given to each of 4 reaction tubes
| + | |
| − | - Two yeast colonie of 4196 and K669 were picked
| + | |
| − | - Incubation at 30°C over night
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| − | 5. Restriction digest of Vector DNA for the transformation
| + | |
| − | - Mix to digest 10 µl DNA of preparation 3.1 for transformation created
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| − |
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| − | Reagent
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| − | Volume for one sample
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| − | Eco RV
| + | |
| − | 1 µl
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| − | Buffer R
| + | |
| − | 5 µl
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| − | Add 20µl H2O
| + | |
| − | 34 µl
| + | |
| − | Σ
| + | |
| − | 40 µl
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| − | - Incubation over night at 37°C
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| − | 6. Overnight culture of YIplac204
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| − | - 5 ml of Ampicilin-media (80µg/ml) wer inoculated with one colony of 1.2 pg 2
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| − | - Incubation at 37°C overnight
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| − | Day 7, 03.07.15
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| − | 1. Transformation with KCPlac22 and KlPlac202 ?????
| + | |
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| − | - over night culture 1:100 with MQ diluted with 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 under flame protection
| + | |
| − | A: 50 ml
| + | |
| − | B: 25 Einheit? → Media wrong estimated
| + | |
| − | - 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 under shaking
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| − | 2. Miniprep with KCPlac204
| + | |
| − | - over night culture (previous day) (6/6) in 2 ml Eppi transferred
| + | |
| − | - centrifugation (700 rpm, 5 min)
| + | |
| − | - 3 times repeated
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| − | - pellet in 90 ml GTE+RNAv transferred
| + | |
| − | - vortex
| + | |
| − | - 100 µl NaOH/SDS Sx inverted
| + | |
| − | - 135 ml KOAC/HoAc transferred Several times inverted
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| − | - 80 µl NaHO/SDS added
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| − | - centrifugation (15 min, 14000 rpm, 8 °C)
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| − | - supernatant in tube transferred
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| − | - in 1 ml 100 % EtOH transferred
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| − | - centrifugation (10 min, 13300 rpm)
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| − | - 1 ml 70 % ethanol added and removed
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| − | - step repeated
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| − | - ethanol dripped down and ?????? at room temperature
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| − | 3. Gelelectrophoresis
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| − | - 1,2 % agarose gel from the rest ?
| + | |
| − | - from over night digestion (6/5) 5 µl given on gel
| + | |
| − | - ?????????????????
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| − | - after 1 h at 120 V gel picture
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| − | - Midiprep from 7/2 used
| + | |
| − | - Scheme marker // Midiprep
| + | |
| − | - after ca. 2 h at 120 V gel picture
| + | |
| − | - ?????????????????
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| − | - Amount of proteins ….
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| − | 4. Transfrmation of the yeast …... G99
| + | |
| − | - 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)
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| − | - pellet in 25 ml ….....
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| − | - pelletised at 3500 rpm and room temperature
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| − | - pellet in 1 ml of 100 mM LiAc resuspended and given in Eppis
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| − | - centrifugation (10 s, 3500 rpm, room temperature)
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| − | - pellet in xxxx 100 mM LiA resuspended on volume of 500 ml
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| − | - for each transformation 50 µl cell suspension (4) in ??????
| + | |
| − | - centrifugated for 10 s and supernatant was discarded/ as well as all probes of 4196 (too less xxxx)
| + | |
| − | - mix of transformation added
| + | |
| − | → 240 µl 50 % PEG 3350
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| − | → 36 µl 1 M LiAc
| + | |
| − | → 5 µl carrierDNA (10mg/ml)
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| − | → 4 µl YEP122 (ex) 5 µl YIP203?????????????? (negative control)
| + | |
| − | → ??? µl of water to YEP122 → 360 µl
| + | |
| − | ?????????????????????? → 360 ml
| + | |
| − | 66 µl ddH2O to YCP22 → 360 µl
| + | |
| − | 64 µl ddH2O to negative control → 360 µl
| + | |
| − | - probe vortexed/ pellets diluted
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| − | - incubation at room temperature (30 °C)
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| − | - heat shock for 20 min at 42 °C
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| − | - centrifugated for 10 s, pellet resuspended in 400 µl H2O
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| − | → YEP122 200 µl plated
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| − | → YEP204 400 µl plated
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| − | → YEP22 200 µl plated
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| − | → negative control 200 µl plated
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| − | - incubated (72 h, 30 °C)
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| − | 5. Precipitation of plasmid-DNA ????????????
| + | |
| − | - to DNA solution xxxx µl NaAC added
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| − | - xxx µl ???? added
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| − | - centrifugation (fullspeed, 5 min, room temperature)
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| − | - DNA appears as a small band
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| − | - remove supernatant
| + | |
| − | - pellet washed in two volumes (240 µl) EtOH 70 %
| + | |
| − | - dried at room temperature for 30 min
| + | |
| − | - DNA solved in 5 µl TE
| + | |
| − | - for experiment 7/4 used
| + | |
| − | | + | |
| − | | + | |
| − | Day 8, 06.07.15
| + | |
| − | | + | |
| − | 1. Examination V7/4
| + | |
| − | - colonies grown!
| + | |
| − | - concept works
| + | |
| − | | + | |
| − | 2. Resuspension of IDT geneBricks xxxxxxxxxxxxx and xxxxxxxxxx
| + | |
| − | | + | |
| − | - 500 ng in 50 µl TE given (c = 10 ng/ml)
| + | |
| − | - incubated (50 °C, 20 min)
| + | |
| − | - vortexed and centrifugated (10 s to fullspeed)
| + | |
| − | | + | |
| − | 3. Restriction diggestion for ligation of xxxxxxxxxxxx and xxxxxxxxx
| + | |
| − | | + | |
| − | - needed amount of DNA
| + | |
| − | → ???????????????
| + | |
| − | → ???????????????
| + | |
| − | → ???????????????
| + | |
| − | | + | |
| − | → ???????????????
| + | |
| − | - following restriction probes were constructed:
| + | |
| − | insert his_spacer_adh1/ insert His_Rep_kl
| + | |
| − | | + | |
| − | DNA
| + | |
| − | 40 µl
| + | |
| − | MM for 3
| + | |
| − | EcoRI
| + | |
| − | 2 µl
| + | |
| − | 6 µl
| + | |
| − | PstI
| + | |
| − | 2 µl
| + | |
| − | 6 µl
| + | |
| − | Buffer0
| + | |
| − | 20 µl
| + | |
| − | 60 µl
| + | |
| − | 200 ddH2O
| + | |
| − | 136 µl
| + | |
| − | 408 µl
| + | |
| − | | + | |
| − | Vector YEOlac181/ YCPlac22/ YEPlac204C7/3
| + | |
| − | DNA
| + | |
| − | 5 µl
| + | |
| − | MM for 4
| + | |
| − | RNAse
| + | |
| − | 1 µl
| + | |
| − | 4 µl
| + | |
| − | EcoRI
| + | |
| − | 1 µl
| + | |
| − | 4 µl
| + | |
| − | PstI
| + | |
| − | 1 µl
| + | |
| − | 4 µl
| + | |
| − | Buffer0
| + | |
| − | 5 µl
| + | |
| − | 20 µl
| + | |
| − | 50 addH2O
| + | |
| − | 37 µl
| + | |
| − | 148 µl
| + | |
| − | | + | |
| − | Vector K801000
| + | |
| − | DNA
| + | |
| − | 40 µl
| + | |
| − | RNAse
| + | |
| − | 1 µl
| + | |
| − | EcoRI
| + | |
| − | 2 µl
| + | |
| − | PstI
| + | |
| − | 2 µl
| + | |
| − | Buffer0
| + | |
| − | 10 µl
| + | |
| − | 100 add H2O
| + | |
| − | 45 µl
| + | |
| − | | + | |
| − | - large probes were chosen because of the high ?????-concentration
| + | |
| − | - over night incubation at 37 °C
| + | |
| − | | + | |
| − | 4. Speedjet PCR-cloning with cccccccccc and xxxxxxxxxxx
| + | |
| − | | + | |
| − | - as a backup the following speedjet probes were generated
| + | |
| − | → 2 µl 10 x ligase buffer
| + | |
| − | → 2,3 µl DNA solution
| + | |
| − | → 1 µl vector
| + | |
| − | → add 19 µl ddH2O → 13,5
| + | |
| − | → 1 µl TKLigase
| + | |
| − | → ??????
| + | |
| − | → incubation (ca. 10 min)
| + | |
| − | | + | |
| − | 5. Transformation
| + | |
| − | | + | |
| − | - Dox-E.colis unfreezed
| + | |
| − | - each 5 µl from 8/4 given on 50 ml competent cells (as positive control ???? on E.coli, as negative control no changes done)
| + | |
| − | - ca 15 min given 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, 07.07.15
| + | |
| − | | + | |
| − | 1. Analysis of over night ?????? from 8/3
| + | |
| − | | + | |
| − | - 1 % agarose gel ??????? (1 g 100 ml THE, 8 µl ???)
| + | |
| − | - each 10 % put on gel with ????? buffer, that means YEP181/ YCPlac22/ YIlac204 → 5 µl
| + | |
| − | K801000 → 10 µl
| + | |
| − | inserts → 20 µl
| + | |
| − | | + | |
| − | - each with 6x ????? buffer xxxxx
| + | |
| − | | + | |
| − | | + | |
| − | 2. Restriction digestion 204
| + | |
| − | | + | |
| − | - 40 µl of plasmid solution 204 EcoRI digested
| + | |
| − | sample from 7/2 S:12
| + | |
| − | → DNA 40 µl
| + | |
| − | EcoRI 2 µl
| + | |
| − | ???? 2 µl
| + | |
| − | Buffer0 20 µl
| + | |
| − | H2O 136 µl
| + | |
| − | → 200 µl
| + | |
| − | - incubation (2h, 37 °C)
| + | |
| − | | + | |
| − | 3. Gels for extraction
| + | |
| − | | + | |
| − | - 1 % gel 100 nl, middle chamber, large comb filled with total digest from 8/3 8µl Roth
| + | |
| − | - 1 % gel 70 ml, small chamber, first comb ????????????
| + | |
| − | | + | |
| − | 4. Gelextraction
| + | |
| − | - middle gel loaded with 45 ml VectorDNA
| + | |
| − | - xxxxxxxxxxxxx
| + | |
| − | - electrophoresis for 2 h at 50 V
| + | |
| − | - ?????????
| + | |
| − | - gel fragment weighted: xxxx mg
| + | |
| − | - 3 time higher volume of QC-buffer added → 1+10 µl QC-buffer added
| + | |
| − | - 10 min at 50 °C gel xxxx
| + | |
| − | - after 3 min and 7 min for 5-7 s vortexed
| + | |
| − | - 450 µl isopropanol added
| + | |
| − | - probe inverted and shortly vortexed
| + | |
| − | - 800 µl on speedcolumn on ??????????
| + | |
| − | - centrifugation (13300 rpm, 1 min) → flow rate discarded
| + | |
| − | - step 3 times repeated
| + | |
| − | - 0,75 ml PE buffer given on column for cleaning
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - column transferred on 1,5 ml tube
| + | |
| − | - 30 µl ??????????? given on column
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - eluate used for further experiments
| + | |
| − | | + | |
| − | 5. Insertcleaning with PcR-Purification-Kit
| + | |
| − | | + | |
| − | - on 180 µl 900 µl PB buffer given
| + | |
| − | - 800 µl probe given on column
| + | |
| − | - column centrifugated (13300 rpm, 1 min) in ??????????????
| + | |
| − | - remaining xxxx µl given on column
| + | |
| − | - column centrifugated at 13300 rpm
| + | |
| − | - both times flow rate was discarded
| + | |
| − | - column loaded with 750 µl PE
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - column transferred on 1,5 ml Eppi
| + | |
| − | - 30 µl BE (elution buffer) given
| + | |
| − | - centrifugation (13300 rpm, 1 min)
| + | |
| − | - eluate used for further experiments
| + | |
| − | | + | |
| − | 6. Controlgel of eluate and digestion of 204 9/2
| + | |
| − | | + | |
| − | - filled on samll from 9/3
| + | |
| − | - ?????:
| + | |
| − | Standard // Vector 22 // Digestion 204 // empty // Standard // Insert of Reporter
| + | |
| − | 6 µl 3 µl 20 µl 6 µl 2,8 µl
| + | |
| − | | + | |
| − | - gelelectrophoresis for 45 min
| + | |
| − | - note: insert was added after 20 min gel current time
| + | |
| − | | + | |
| − | 7. ÜNR
| + | |
| − | | + | |
| − | - 2 colonies on plate 204, Pjet_His_spacer, Pjet_reporter_klein picked
| + | |
| − | - to each 5 ml 80 µg/µl ampiciline medium given
| + | |
| − | - over night incubation at 37 °C
| + | |
| − | | + | |
| − | | + | |
| − | Day 10, 08.07.15
| + | |
| − | | + | |
| − | 1. Ligation of the linearised vector VCPlac22 xxxxxxxxxxxxxxx
| + | |
| − | | + | |
| − | - 3 probes for diligation generated → ligation with insert
| + | |
| − | - 3 µl vector (YCPlac22) linearized
| + | |
| − | - 14 µl insert (His3 spacer) linearized
| + | |
| − | - 2 µl ligase buffer
| + | |
| − | - 1 µl T4 Ligase
| + | |
| − | → Re-Ligation control without insert
| + | |
| − | | + | |
| − | - 3 µl vector (YCPlac22) linearized
| + | |
| − | - 14 µl add H2O
| + | |
| − | - 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 for 3 h at room temperature incubated
| + | |
| − | | + | |
| − | 2. Miniprep from (9/7 S 20)
| + | |
| − | | + | |
| − | - 2 ml from over night culture in 2 ml-Eppi transferred
| + | |
| − | - centrifugation (7000 rpm, 5 min), supernatant ????
| + | |
| − | - 2 ml from over night culture in appropriate Eppis from step 1 transferrred
| + | |
| − | - centrifugation (7000 rpm, 5 min), übereinander, supernatant ????
| + | |
| − | - pellet in 250 µl xxx resuspended
| + | |
| − | - 250 µl buffer P2 added
| + | |
| − | - 5 times inverted
| + | |
| − | - 350 µl buffer N3 added
| + | |
| − | - 5 times inverted
| + | |
| − | - cemtrifugation (13000 rpm, 10 min)
| + | |
| − | - supernatant loaded on column
| + | |
| − | - centrifugation (13000 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - 750 µl PE buffer added
| + | |
| − | - centrifugation (13000 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - centrifugation (13000 rpm, 1 min)
| + | |
| − | - flow rate discarded
| + | |
| − | - columns loaded in new Eppis
| + | |
| − | - 50 µl elution buffer added
| + | |
| − | - 1 min resting
| + | |
| − | - centrifugation (13000 rpm, 1 min)
| + | |
| − | | + | |
| − | 3. Digestion of Miniprep
| + | |
| − | | + | |
| − | - ???????????
| + | |
| − | → 1 µl DNA xxxxxxxxxxxxxx → MM for 7 probes for each 19 µl
| + | |
| − | 0,5 µl EcoRI 3,5 µl EcoRI
| + | |
| − | 0,5 µl PstI 3,3 µl PstI
| + | |
| − | 2 µl Buffer0 14 µl Buffer0
| + | |
| − | 16 µl ddH2O 112 µl ddH2O
| + | |
| − | - digestion for 3 h at 37 °C
| + | |
| − | | + | |
| − | | + | |
| − | 7. Gelelectrophoresis of the Digestion from 3
| + | |
| − | - 20 µl from digestion taken and with xxxxxxxxxxxxxx
| + | |
| − | - gelelectrophoresis (1 h, 120 V)
| + | |
| − | - ???????????
| + | |
| − | ???????????
| + | |
| − | ?????????ß?
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | Day 11, 09.07.15
| + | |
| − | | + | |
| − | 1. Culture picking for over night culture
| + | |
| − | | + | |
| − | - 12 colonies from 100 µl xxxxx taken (compare S2310/6) with pipette peak
| + | |
| − | - in 2 ml 80 µg/ml ampiciline medium given
| + | |
| − | - 2 colonies picked from YFP-plate
| + | |
| − | - 5 ml 25 µg/ml Cm medium inoculated
| + | |
| − | - over night incubation at 37 °C
| + | |
| − | | + | |
| − | | + | |
| − | Day 12, 10.07.15
| + | |
| − | | + | |
| − | 1. Miniprep of the over night culture from 11/1
| + | |
| − | | + | |
| − | - over night culture transferred in 2 ml Eppi
| + | |
| − | - centrifugation (7000 rpm, 5 min)
| + | |
| − | - ????????????????
| + | |
| − | - supernatant discarded
| + | |
| − | - performed like 3/1 S 3
| + | |
| − | - ????????????
| + | |
| − | | + | |
| − | 2. Restriction digestion
| + | |
| − | - ??????????? for all Minipreps (14)
| + | |
| − | → 3 µl DNA-solution MM for 15
| + | |
| − | 0,5 µl EcoRI 7,5 µl EcoRI
| + | |
| − | 0,5 µl PstI 7,5 µl PstI
| + | |
| − | 2 µl Buffer0 30 µl Buffer0
| + | |
| − | 14 µl ddH2O 210 µl ddH2O
| + | |
| − | - incubation (1,5 h, 37 °C)
| + | |
| − | | + | |
| − | 3. Gelelectrophoresis
| + | |
| − | - 2 gels (one with 100 ml and one with 70 ml) ????
| + | |
| − | - 13,6 µl Roth Ethidiumbromide added
| + | |
| − | - probes from 12/2 with 4 µl ???????????????????????
| + | |
| − | - ????? as followed:
| + | |
| − | → Gel I: 5 x empty // standard // YCPlac22 + insert 1 //
| + | |
| − | 2 //
| + | |
| − | 3 //
| + | |
| − | 4 //
| + | |
| − | 5 //
| + | |
| − | 6 //
| + | |
| − | 7 //
| + | |
| − | 8 //
| + | |
| − | | + | |
| − | → Gel II: standard // YCPlac22 + insert 9 //
| + | |
| − | 10 //
| + | |
| − | 11 //
| + | |
| − | 12 //
| + | |
| − | empty //
| + | |
| − | YFP 1 //
| + | |
| − | YFP 2 //
| + | |
| − | stamdard //
| + | |
| − | - gelelectrophoresis for 1 h
| + | |
| − | - over night culture ????????????????
| + | |
| − | | + | |
| − | | + | |
| − | Day 13, 13.07.15
| + | |
| − | | + | |
| − | 1. Miniprep via Quiagen column
| + | |
| − | | + | |
| − | - 5 ml over night culture from 12/3 centrifuged (7000 rpm, 5min) in 2 ml Eppis ????????
| + | |
| − | - Quiagen Miniprep analogue to 10/2 performed (see page 21)
| + | |
| − | | + | |
| − | | + | |
| − | 2. Restriction digestion
| + | |
| − | - ???? probes from 13/1 (see above) EcoRI PstI digested Satz komisch
| + | |
| − | - MM created
| + | |
| − | → single Mastermix for 13 probes
| + | |
| − | 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)
| + | |
| − | | + | |
| − | 3. Gelelectrophoresis
| + | |
| − | | + | |
| − | - one 1 % agarose gel 100 ml moulded
| + | |
| − | - after cooling 8 µl ethidium bromide added
| + | |
| − | - 14 ??????? used
| + | |
| − | - to 12 probes from 13/2 each 4 µl ????????? added
| + | |
| − | - 24 µl loaded on gel according to following scheme
| + | |
| − | standard //
| + | |
| − | YCPlac 22 + insert 1 //
| + | |
| − | YCPlac 22 + insert 2 //
| + | |
| − | YCPlac 22 + insert 3 //
| + | |
| − | YCPlac 22 + insert 4 //
| + | |
| − | ...
| + | |
| − | YCPlac 22 + insert 12 //
| + | |
| − | 4814 for IS
| + | |
| − | - gelelectrophoresis (1 h, 120 V)
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | Day 14, 14.07.15
| + | |
| − | | + | |
| − | 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)
| + | |
| − | | + | |
| − | 2. Addition of restriction sites via PCR
| + | |
| − | | + | |
| − | | + | |
| − | probe
| + | |
| − | water control
| + | |
| − | H2O
| + | |
| − | 71 µl
| + | |
| − | 73 µl
| + | |
| − | 5 x buffer
| + | |
| − | 20 µl
| + | |
| − | 20 µl
| + | |
| − | template
| + | |
| − | -
| + | |
| − | 2 µl
| + | |
| − | ????
| + | |
| − | 2 µl
| + | |
| − | 2 µl
| + | |
| − | revPrimer
| + | |
| − | 2 µl
| + | |
| − | 2 µl
| + | |
| − | ????
| + | |
| − | 1 µl
| + | |
| − | 1 µl
| + | |
| − | | + | |
| − | Σ 100 µl
| + | |
| − | Σ 100 µl → next time less
| + | |
| − | - 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
| + | |
| − | | + | |
| − | | + | |
| − | 3. Gelelectrophoresis of the restriction digestion and the PCR
| + | |
| − | | + | |
| − | - 70 ml 1 % agarose gel moulded
| + | |
| − | - after cooling 8 µl ethidium bromide added
| + | |
| − | - from PCR 5 µl with 1 µl ??????????????
| + | |
| − | - to digestion 4 µl ????? added
| + | |
| − | - according to following scheme loaded
| + | |
| − | → standard – digestion – water control – PCR
| + | |
| − | - gelelectrophoresis (1 h, 120 V)
| + | |
| − | | + | |
| − | | + | |
| − | 4. Purification of the PCR fragment
| + | |
| − | | + | |
| − | - analogue to 9/5 page 19
| + | |
| − | - pellet was solved in 30 µl EB
| + | |
| − | | + | |
| − | | + | |
| − | 5. Restriction digestion ofthe YFP-PCR 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))
| + | |
| − | | + | |
| − | | + | |
| − | 6. Gelextraction of the digested vector
| + | |
| − | | + | |
| − | - gelelectrophoresis (1,5 h) in 1 % agarose gel analogue 9/4 page 19
| + | |
| − | - band with scalpel removed and weighted = 390 mg
| + | |
| − | - three fold amount of QC added (1160 µl)
| + | |
| − | - analogue to 9/4 page 19 conducted
| + | |
| − | | + | |
| − | | + | |
| − | Day 15, 15.07.15
| + | |
| − | | + | |
| − | 1. Purification of the digestion from 14/5
| + | |
| − | | + | |
| − | - analogue to 9/5 page 19
| + | |
| − | - 380 µl PB used
| + | |
| − | - in 30 µl solved da fehlt iwas
| + | |
| − | | + | |
| − | 2. Dephosphorylation of the vector
| + | |
| − | | + | |
| − | - alkaline phosphatase mix created
| + | |
| − | → 20 µl TCPlac22 + insert XhoI Sal I digested iwas fehlt
| + | |
| − | 3 µl FAP buffer
| + | |
| − | 1 µl FAP
| + | |
| − | 6 µl H2O
| + | |
| − | - incubation (20 min, 37 °C)
| + | |
| − | - incubation (5 min, 75 °C)
| + | |
| − | | + | |
| − | 3. Ligation YCPlac22 + insert ????? and YFP
| + | |
| − | | + | |
| − | - 3 liagtionansätze prepared
| + | |
| − | | + | |
| − | A) ??? µl vector, dephosphorylated (YCPlac22)
| + | |
| − | 5 µl insert (YFP)
| + | |
| − | 2 µl ligase buffer
| + | |
| − | 1 µl T4 ligase
| + | |
| − | 8 µl ddH2O
| + | |
| − | | + | |
| − | B) ??? 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)
| + | |
| − | | + | |
| − | | + | |
| − | 4. Transformation of the ligation
| + | |
| − | | + | |
| − | - analogue to 8/5 conducted
| + | |
| − | - transformation as followed plated
| + | |
| − | 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 ??? and H2O on ampiciline plated
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | Day 16, 16.07.15
| + | |
| − | | + | |
| − | 1. ??????
| + | |
| − | - Bleistift
| + | |
| − | - 20 colonies of the colonies picked and in each 5 m 100 µg/ml ampiciline medium given da passt iwas nicht → over night culture at 37 °C
| + | |
| − | | + | |
| − | 2. Trafo from 15/3 repeated
| + | |
| − | | + | |
| − | - anologue to 15/3 conducted
| + | |
| − | | + | |
| − | | + | |
| − | Day 17, 17.07.15
| + | |
| − | | + | |
| − | 1. Miniprep of the over night culture
| + | |
| − | | + | |
| − | - anologue to 3/1 conducted
| + | |
| − | | + | |
| − | 2. Restriction digestion from the Miniprep
| + | |
| − | | + | |
| − | - restriction ??????? created
| + | |
| − | → 1 µl Miniprep MM for 21
| + | |
| − | 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, ??? °C)
| + | |
| − | | + | |
| − | 3. Gelelectrophoresis
| + | |
| − | - each probe with 4 µl ??????
| + | |
| − | - ??? µl ????
| + | |
| − | - 6 µl standard loaded
| + | |
| − | - scheme: standard // Miniprep 1 – 10 = Gel I
| + | |
| − | standard // Miniprep 11 – 20 = Gel II
| + | |
| − | - expected fragments ~ 5000 bp
| + | |
| − | ~ 700 bp
| + | |
| − | | + | |
| − | | + | |
| | </html> | | </html> |
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