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
  • 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

  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 (6µl) // YCplac22 (3µl) //YIplac204 digested (20µl) // empty // Standard (6µl) // Insert his3_reporter_klein (2.8µl)
  • 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 DNA
• concentration is still above the 25ng per µl. Detection of the Insert his_rep_klein was possible.

  7. Overnight culture of Pjet-transformed 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:

  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

  2. Miniprep from overnight culture day 9/7.

  • 2 ml over night culture transferred to 2 ml
  • reaction tubes
  • centrifugation (7000 rpm, 5 min)
  • supernatant discarded
  • 2 ml from over night culture transferred in the same reaction tubes as their precursors to increase the amount of bacteria
  • centrifugation (7000 rpm, 5 min)
  • supernatant discarded
  • DNA
  • Preparation conducted as given by Quia
  • Gen Miniprep instruction
  • Elution in 50 µl elution buffer

  3. Digestion of Miniprep

  • Digest prepared

  	1 µl DNA from the miniprep(see above)	MM for 7 samples à 19 µl
  EcoRI	0.5 µl	3.5 µl
  PstI	0.5 µl	3.5 µl
  Buffer O	2 µl	14 µl
  ddH2O	16 µl	112 µl

  • digestion for 3 h at 37 °C

  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

  5 Preparation of X

  gal plates

  • 40µl 2% x-gal spread on 6 ampicilin enriched plates
  • 40µl IPTG spread on the dried plates
  • 40µl dH2O spread on the dried plates

  6 Transformation of Ligation and EYFP (BBa_E2030)

  • Each of folowing DNA
  • samples added to 50 µl DH5α on ice
    • 5µl ligation from 10.1
    • 5µl ligase + vectorligation from 10.1
    • 5µl vector + ligase buffer solution from 10.1
    • 1µl pBluescript
    • 5µl H2O
    • 2.5µl EYFP (BBa_E2030)
  • Transformation analogous to 8.5
  • Transformed E. coli samples spread on ampiciline plates as followed
    • 100 µl ligation
    • 200 µl ligation
    • 200 µl ligase + vector
    • 200 µl pBluescript
    • 200 µl H2O
  • Remaining transformed bacteria spread on chloramphenicole agar plates
    • 200 µl EYFP (BBa_E2030)
    • 200 µl H2O

  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:
  &rArr his3_rep_klein A // his3_rep_klein B // his_spacer_adh1 A // his_spacer_adh1 B// YIplac204 A //YIplac 204 B// 1kb DNA-marker Foto: 150708a.png http://www.studon.unierlangen.de/studon/ilias.php?ref_id=1325854&cmd=return&cmdClass=ilrepositorygui&cmdNode=o8&baseClass=ilRepositoryGUI&redirectSource=ilobjfilegui&cmdMode= 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 YFP
  • plate
  • 5 ml 25 µg/ml Canamycin medium inoculated
  • over night incubation at 37 °C

  Day 12, 15/07/10

  1. Miniprep of the over night culture from day 11/1. (see above)

  • conducted according to protocol 1
  • eluted in 30µl 1x TE

  2. Restriction digestion

  • Restriction for all DNA-preparations (14 samples)

  DNA Solution	3 µl	MM for 15
  PstI	0.5 µl	7.5 µl
  EcoRI	0.5 µl	7.5 µl
  Buffer O	2 µl	30 µl
  ddH2O	14 µl	210 µl

  • incubation (1.5 h, 37 °C)

  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 DNA-marker //	YCPlac22 + insert 1 //
  		2 //
  		3 //
  		4 //
  		5 //
  		6 //
  		7 //
  		8 //

  Gel II:	1kb DNA-marker // YCPlac22 + insert	9 //
  		10 //
  		11 //
  		12 //
  		empty//
  		YFP clone 1 //
  		YFP clone 2//
  		stamdard//

  • Results not expected ratio insert vector seems tilted
  • Over night culture of all samples analogous to Day 11 1.
  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

  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

  2. Restriction digestion

  • DNA-Solutions obtained in Day 13/1. (see above) EcoRI PstI digested
  • Mastermix created

  single sample	Mastermix for 13 Samples
  1 µl DNA	-
  0.5 µl EcoRI	6.5 µl EcoRI
  0.5 µl PstI	6.5 µl PstI
  2 µl Buffer0	26 µl Buffer0
  16 µl ddH2O	208 µl ddH2O

  • incubation (2 h, 37 °C)

  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 DNA-marker //
    • 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

    1. Restriction digestion XhoI, Sal I from YCPlac22 + insert10 (day 13/13)

    • restriction digestion conducted

      DNA YCPlac22 + insert10 from day 13	20 µl
      Sal I	2 µl
      XhoI	4 µl
      Buffer 0	10 µl
      ddH2O	64 µl

      • incubation (2 h, 37 °C)

      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

      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 PCR
    • sample
    • 2 µl staining solution added to 10 µl of digest
    • According to following scheme loaded 1kb DNA
    • marker // 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

      4. Purification of the PCR fragment

      • Analogous to Day 9/5.
      • pellet was solved in 30 µl EB

      5. Restriction digestion of the YFP-PCR fragment

      • purified insert digested with XhoI Sal I

      DNA	30 µl
      Sal I	2 µl
      XhoI	4 µl
      Buffer O	10 µl
      ddH2O	54 µl

      • incubation (3 h, 37 °C))

      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

      1. Purification of the digestion from day 14/5.

      • Analogue to Day 9/5.
      • Changes to Day 9/5.
        • 380 µl PB used
        • eluted in 30 µl Elution Buffer

        2. Dephosphorylation of the vector

        • alkaline phosphatase mix created

        TCPlac22 + insert XhoI Sal I digested	20 µl
        FAP buffer	3 µl
        FAP	1 µl
        H2O	6 µl

        • incubation (20 min, 37 °C)
        • incubation (5 min, 75 °C)

        3. Ligation 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)

        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 PBSC-Bluescript and H2O on ampiciline plated

        Day 16, 15/07/16

        1. Picking of colonies

        • 20 colonies of the ligation plate picked
        • Each given in 5 ml 100 µg/ml ampiciline medium
        • Incubation over night at 37 °C

        2. Retransformation of the ligation

        • Repition of Day 15 3. (see above)

        Day 17, 15/07/17

        1. Miniprep of the over night culture

        • Performed according to protocol 1

        2. Restriction digestion from the Miniprep

        • restriction digest created

        Miniprep	1 µl	Mastermix for 21 samples
        Sal I	0.5 µl	10.5 µl
        XhoI	1 µl	21 µl
        Buffer 0	2 µl	42 µl
        ddH2O	15.5 µl	325.5 µl

        
        
        • digestion (2 h, 37 °C)

        3. Gelelectrophoresis

        • Gelelectrophoresis performed according to protocol 2
        • 4 µl staining solution was added to each digest
        • 6 µl 1kb DNA-marker loaded
        • scheme:

          kb DNA-marker // Miniprep 1 - 10 = Gel I	1kb DNA-marker // 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 XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not. b)GelII Restriction sites XhoI and SalI are compatible, but after XhoI-SalI-ligation the restriction site is removed. Thus "homo"-ligation creates an insert after XhoI-SalI digestion whereas "hetero"-ligation does not.
          • Clones 1, 8, 9, 12, 14, 18 had an insert, which were similar to the expected size

          Day 18, 15/07/20

          1. Overnight culture of S. cerevisiae K699 and 4196

          • Two colonies picked of each strain
          • Inoculation of 5 ml YEPD-Medium each
          • Incubation overnight at 28°C

          Day 19, 15/07/21

          1. Transformation of S. cerevisiae strains K699 and 4196 with YCplac22-YFP

          • over night culture dilitued 1:100 with MQ (10 µl suspension and 990 µl MQ)
          • measurement 0D600:

          699 A =	0.019	→ used
          699 B =	0.014
          4196 A =	0.108	→ used
          4196 B=	0.056

        • two flasks filled with YEPG next to Bunsenburner

          A: 25 ml

          B: 25 ml

          • 4,46 ml added to flask A suspension 699 oD600 = 0,208
          • 0.58ml added to flask B suspension 4196 oD600 = 0,193
          • 3 h at 30 °C and incubated while shaking
          • 25 ml yeast culture from the flask given in 50 ml falcon
          • oD600 determined

          699 = 0.543

          4196 = 0.503

          • Centrifugation (5 min, 3500 rpm, room temperature)
          • Supernatant discarded
          • Contamination in 4196 suspension detected => Sample discarded
          • Pellet resuspended in 25 ml ddH2O
          • Pelletised at 3500 rpm and room temperature
          • Supernatant discarded
          • Pellet in 1 ml of 100 mM LiAc resuspended and given in 1.5 reaction tubes
          • Centrifugation (10 s, 3500 rpm, room temperature)
          • Supernatant discarded
          • Pellet resuspended in 500 µl 100 mM LiAc
          • For each transformation 50 µl cell suspension (6) transferred in 1.5ml ml reaction tubes
          • Centrifugated for 10 s and supernatant discarded
          • Mix for transformation added (adhered to order as written below)
            • > 240 µl 50 % PEG 3350
            • > 36 µl 1 M LiAc
            • > 5 µl carrier DNA (10mg/ml)
            • > 5µl ddH2O (negative control)/ YCplac22-YFP 1, 8, 9, 12, 14 and 18
            • > 64 µl of ddH2O
          • Sample vortexed until pellet was resuspended
          • Incubation at room temperature (30 °C)
          • Heat shock for 20 min at 42 °C
          • Centrifugated for 10 s,
          • Pellet resuspended in 400 µl H2O
          • 200 µl and 100µl of transformation plated on mediaplates without tryptophan
          • Incubated (72 h, 30 °C)

          Day 20, 15/07/24

          1. Convokal Microskopy

          • One colony per construct picked and diluted in Water
          • Microscopy → see result section

          1 von Adrian

          1 von Filip

          1 von Vlady (schon auf Studon)

          1 von Frederike