Team:Potsdam/Notebook/Highcopy

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Lab book

We separated our lab book into the following parts:



Wednesday, 07/26/17

-Digesting with
Enzyme Master Mix for Plasmid Backbones

5 ul Cut Smart Buffer
18,5 ul dH2O
0,5 ul EcoR1-HF (restriction enzyme)
0,5 ul Pst1 (restriction enzyme)
0,5 ul Dpn1 (restriction enzyme)
Digest Plasmid Backbone
4 ul Master Mix
4 ul Fragment
approach 1: psb1a3
approach 2: psb1c3
approach 3: psb1k3
incubate 60 min at 37°C
heat kill (20 min) at 80°C
Digesting with
20 ul Fragment (IDT: Scaffold 2-12, c = 10 ng/ul)
1ul EcoR1-HF (restriction enzyme)
1 ul Pst1 (restriction enzyme)
4 ul Cut Smart Buffer
17 ul dH2O
incubate 60 min at 37°C
heat kill (20 min) at 80°C


Thursday, 08/09/17

prepared plates
1l Media with Agar

chemical mass in g
Trypton 10
yeast extract
 5
NaCl 5
Agar 15


500 ml with AMP
500 ml with CAP

-resuspended with 100ul TE-Buffer:
Taget cassett 1 (Scafffold 1-6), IDT
Taget cassett 2 (Scafffold 2-12), IDT
Taget cassett 3 (Scafffold 3-18), IDT
IAAM
LacI
IAAH

  • decant "10x Buffer for T4 DNA Ligase with 10mM ATP"

    • 10 PCR-Tubes, each with 50 µl

  • Digestion of the three target cassetts (Digestion Protocoll)

    • nomenclature

    • tube 1: VIG_C_TaC_1_D_EcoR1_Pst1; Taget cassett 1 (Scafffold 1-6)
    • tube 2: VIG_C_TaC_2_D_EcoR1_Pst1; Taget cassett 2 (Scafffold 2-12)
    • tube 3: VIG_C_TaC_3_D_EcoR1_Pst1; Taget cassett 3 (Scafffold 3-18)
    tube 1 tube 2 tube 3
    10xNEB-Buffer 5 ul 5 ul 5 ul
    EcoR1-HF 1 ul 1 ul 1 ul
    Pst1 1 ul 1 ul 1 ul
    Water 23 ul 23 ul 23 ul
    Target cassett 1 10 ul - -
    Target cassett 2 - 20 ul -
    Target cassett 3 - - 20 ul
    • the enzymes are added last

    • 1 hour incubation at 37°C

    • 20 minutes heat kill at 80°C

  • Ligation of the Vectors and Inserts

    • nomenclature

    • Backbones (digested)

    • psB1C3_D: IG_C_PBa_CAP_D_EcoR1_Pst1

    • psB1A3_D: IG_C_PBa_A_D_EcoR1_Pst1

    • psB1K3_D: IG_C_PBa_K_D_EcoR1_Pst1

    • Inserts = taget cassetts (digested)

    • TaC_1_D: IG_C_TaC_1_D_EcoR1_Pst1; Taget cassett 1 (Scafffold 1-6)

    • TaC_2_D: IG_C_TaC_2_D_EcoR1_Pst1; Taget cassett 2 (Scafffold 2-12)

    • TaC_3_D: IG_C_TaC_3_D_EcoR1_Pst1; Taget cassett 3 (Scafffold 3-18)

    Eppi Plasmid Backbone (2ul) Target cassett (8,8ul) T4 Ligase 10x Ligase Buffer Water
    1 psB1C3_D TaC_1_D 1 µl 2 µl 6,2 µl
    2 psB1C3_D TaC_2_D 1 µl 2 µl 6,2 µl
    3 psB1C3_D TaC_3_D 1 µl 2 µl 6,2 µl
    4 psB1A3_D TaC_1_D 1 µl 2 µl
    		 6,2 µl
    5 psB1A3_D TaC_2_D 1 µl 2 µl 6,2 µl
    6 psB1A3_D TaC_3_D 1 µl 2 µl 6,2 µl
    7 psB1K3_D TaC_1_D 1 µl 2 µl 6,2 µl
    8 psB1K3_D TaC_2_D 1 µl 2 µl 6,2 µl
    9 psB1K3_D TaC_3_D 1 µl 2 µl
    		 6,2 µl
    • the enzymes are added at least
    • 10 minutes incubation at room temperature
    • 10 minutes heat kill at 65°C


    Thursday, 08/10/17

  • transformed ligated target cassettes of yesterday on corresponding antibiotics
  • cassettes transformed:
    • IG_C_CAP_TAC1, IG_C_CAP_TAC2, IG_C_CAP_TAC3
    • IG_C_K_TAC1, IG_C_K_TAC2, IG_C_K_TAC3
    • IG_C_A_TAC1, IG_C_A_TAC2, IG_C_A_TAC3


    Friday, 08/11/17

  • transformed ligated target cassettes the same way as at the 10.08.17, because there where no colonies in the plates, we used the competent E.coli-cells from Fabian for this transformation, only difference from the protocoll: we used just 450 µl LB-media after transformation instead of 900 µl

  • To do for Monday: Prepare 500 ml 1% [w/v] agarose (5 g agarose in 500 ml 1 x TAE buffer)


    • Monday, 08/14/17

  • Colony PCR with transformants from the 10th of August
  • prepared 500 mL of Agarose buffer
  • diluted primers from 100 µM to 10 µM
  • prepared master mix with:
    • dH2O 262,5 µL
    Taq-Polymerase (All in Red Taq) 312,5 µL
    Primer 1 (10 µM) - IG_C_Q_17 25 µL
    Primer 2 (10 µM) - IG_C_Q_18 25 µL
  • Added 10 µL of the mastermix into the prepared tubes (51 tubes: 6 colonies per plate chosen, but on one only 3)
  • picked colonies from Trafo-plate (10.08.), dipped once on master plates and then incubated tips in tubes
  • Master plates are in the fridge
  • No negative controlls were done!
  • Cycling conditions:
    • 60 sec 95°
    15 sec 95° 35 x
    15 sec 52° -> annealing temperature: probably not suitable due to bad results
    20 sec 72°
    5 min 72°
  • Gel electrophoresis: no loading buffer added (included in Red Taq master mix)
  • Loaded 10 µL per slot (~whole tube)
  • gel picture in the Lab book -> colonie 7, 15,16 and 50 might carry the insert
  • Prepared 10 µL preculuture with the four clones that showed to have the insert (successful transformation)
    -> shaking at 37° overnight
  • Prepared LB media with CAP and Kan (we have Carb, which is similar to Amp)


    • Tuesday, 08/15/17

  • *repeated colony PCR with transformants from the 10th of August


  • *nomenclature:
    • KAN + Tac3: 1 to 8
    KAN + Tac2: 9 to 16
    KAN + Tac1: 17 to 24
    AMP + Tac1: 25 to 32
    AMP + Tac2: 33 to 40
    AMP + Tac3: 41 to 48
    CAP + Tac1: 49 to 54
    CAP + Tac2: 55 to 57

  • *Mastermix:
  • used the same volume as yesterday (14/08/17)
  • prepared Mastermix and
  • Added 10 µL of the mastermix into the prepared tubes (57 tubes: 8 colonies per plate chosen, but on one only 3)
  • picked colonies from Trafo-plate (10.08.), dipped once on master plates and then incubated tips in tubes

  • *Cycling condititons:
  • same as yesterday, but used 50°C as Annealing Temperature
  • program: iGEM1 in PeQlab thermocycler

  • run gel at 140V and 30 min
  • results worse than yesterday

  • prepared liquid culture (AMP, CAP, KAN with the Tac1/2/3)


    • Wednesday, 08/16/17

  • digested target-cassettes and backbones again
  • prepared 50 uL 5x restriction Mastermix for backbones:
    • NEBuffer 2.1: 10 µL
    • EcoRI: 2,5 µL
    • PstI: 2,5 µL
    • dpnI: 2,5 µL
    • ddH2O: 32,5 µL
  • digested 10 µL (250 ng) ackbone with 10 µL Mastermix of the plasmids pSB1C3, pSB1K and pSB1A3
  • labeled tubes as in the to-do list and put date AND on every new tube (also for cassette digest) a green dot on the Eppi to how that it's the new one!
  • all put into high-copy box in -20 and used for ligation later
  • digested target cassettes (TaC) with EcoRI and PstI:
    • DNA: 40 µL (400 ng)
    • NEBuffer 2.1: 5 µL
    • EcoRI: 1 µL
    • PstI: 1 µL
    • ddH2O: 3 µL
  • labeled tubes like in to-do list and put gren dot on them, put into high-copy box in -20 and used for ligation both digests were prepared and incubated in 37°C for 1:20 h and then heat killed for 20 min at 80°C
  • ligations of backbones and cassettes:
    • Eppi full name Volume plasmid Volume cassette T4 ligase T4 buffer H20
    C1 CAP_Tac1 4 µL 8,2 µl
    		 1 µL 2 µL 4,8 µl
    C2 CAP_Tac2 4 µL 8,6 µl
    		 1 µL 2 µL 4,4 µl
    C3 CAP_Tac3 4 µL 9,1 µl
    		 1 µL 2 µL 3,9 µl
    A1 A_Tac1 4 µL 7,9 µl
    		 1 µL 2 µL 5,1 µl
    A2 A_Tac2 4 µL 8,3 µl
    		 1 µL 2 µL 4,7 µl
    A3 A_Tac3 4 µL 8,7 µl
    		 1 µL 2 µL 4,3 µl
    K1 K_Tac1 4 µL 7,7 µl
    		 1 µL 2 µL 5,3 µl
    K2 K_Tac2 4 µL 8,1 µl
    		 1 µL 2 µL 4,9 µl
    K3 K_Tac3 4 µL 8,5 µl
    		 1 µL 2 µL 4,5 µl
  • ligated over night at 16°C, tomorrow transformation
  • ran all digested fragmentsd in gel to check for size, not always enough DNA in the pockets but 3 bands at the right size could be seen


    • Thursday, 08/17/17

    Transformation of ligation reaction from 16.08 in E.coli cells ( as competent cells, our cells)
  • Transformation reaction according to the Transformation protocol
  • only difference from the protocol at 8. used 950 uL of 2xYT- Medium from Fabian instead of SOC-Medium !!
  • nomenclature:
    • Trafo TaC 1 200 µL
    • Trafo TaC 2 200 µL
    • Trafo TaC 3 200 µL
    • Trafo TaC 1 concentrated
    • Trafo TaC 2 concentrated
    • Trafo TaC 3 concentrated --> for each antibiotic Cap, Kan, Amp 6 plates
  • concentrated means after spin down of the whole transformation mixture,removal of nearly all supernatant and resuspension
    • -> incubated 18 plates overnight at 37°
  • prepared plates Kan, Cap
    • 1L LB- Media with Agar:
        • - 10 g Trypton
        - 5g yeast extract
        - 5g NaCl
        - 15g Agar
    • added the antibiotica right before pouring the plates
    • for 500 ml LB -Agar + 500 µL antibiotica
  • -> result plates with Kan, Cap stored in the freezer room


    • Friday, 08/18/17


  • Transformation of NEB 5-alpha Competent E.coli (C298) with ligated target cassettes
  • after adding SOC-Media incubate at 37°C
    • 20 min with 300 rpm
    • 40 min with 800 rpm


  • SOC Media:
    • mix with 270ml dH2O:
      • 6g Bacto Tryptone
      • 1,5g Bacto Yeast Extract
      • 0,6ml 5M NaCl
      • 0,75ml 1M KCl
      • 3ml 1M MgCl2
      • 3ml 1M MgSO4
    • padding to 295 ml with dH2O and autoclave
    • 1M glucose (sterile filtration)
    • 10ml dH2O
    • 1,8g glucose


    Monday, 08/21/17

  • LB-plates with Trafo from 18.08.17:
    • plates with 200 µL Transformation solution show no or few colonies
    • plates with concentrated transforation show more colonies
      • --> except AmpTAC2(no colonies) and AmpTAC3(1 colony)
    • for colony CR 6 clones are chosen from each plate (except AMP TAC3--> only 1 clone and AMP TAC3--> no clone)
    • for control: BBa_K314110 (Kit plate 1 Well 1A)--> estimated PCR product length:749 bp)
    Preparation of Masternix (Volume for 50 reactions)
      dH2O 210 µL All in TM Red Taq 250 µL IG_C_Q17 (10 µM) 20 µL IG_C_Q18 (10 µM) 20 µL --> 43 aliquotes á 10 µL
  • Pick clone--> transfer on Maserplate--> resuspend in 10 µL Mastermix in the corresponding tubes
    • tubes:
      1-6 CAPTAC1 7-12 CAPTAC2 13-18 CAPTAC3 19-24 KANTAC1 25-30 KANTAC2 31-36 KANTAC3 37-42 AMPTAC1 43 AMPTAC3 44--> control
    --> for control resuspend plasmid in Well 1A with 10 µL H2O --> preparation of PCR sample:
      dH2O 7 µL All in TM Red Taq 12,5 µL IG_C_Q17 0,4 µL IG_C_Q18 0,4 µL resuspended plasmid 10 µL
    --> flick tubes--> spin shortly down --> start PCR program (slightly changed) "iGEM2" using Mastercycler (not torsten the Thermocycler)
    initial Denaturation 4min 95°C
    Denaturation 15s 95°C x 35
    Annealing 15s 52°C
    Elongation 30s 72°C
    final Elongation 5 min 72°C
  • prepare 1% Agarose-gel
  • load samples and 1kb marker on gel
  • run for 25 min at 140V

    • --> all clones of CAPTAC1 show right bands(1,2,3,4,5,6)
    --> clones 19,21,22,23 and 24 of KANTAC1 show right bands
    --> clones 38,39 of AMPTAC1 show right band
    --> clone 17 of CAPTAC3 show right band
  • All clones (except clone 17) which should contain plasmids containing TAC2 or TAC3 dont show right band

    • --> 5 mL liquid cultures of clones 2,6,17,22,23,38 ans 39 are prepared (conteining the corresponding anitibiotic 5 µL)
    --> incubate shaking at 37°C over night


    Wednesday, 08/23/17

    test digested again, succesfull for all tested colonies:
    • A38/39: TaC1 in Amp
    • C2/6: Tac1 in CAP
    • C17: Tac3 in Cap
    • K22/23: Tac1 in Kan
    A38, C2, c17 and K22 were sent for seuqencing:
    Sequencing ID
    		 Plasmid/primer combination
    33FE06 A38/Q17
    33FE07 A38/Q18
    33FE08 C2/Q17
    33FE09 C2/Q18
    33FE10 C17/Q17
    33FE11 C17/Q18
    33FE12 K22/Q17
    33FE13 K22/Q18
  • for sequencing: plasmid was diluted to 100ng/µL and primer to 5 µM
  • 5 µL Primer + 5 µL Plasmid
  • PCR for amplification of IDT fragments, see low-copy lab book (page 35 in the real one)
  • Eppis: TaC1, TaC2, TaC3
  • Primer: Q31/32, Q33/34, Q33/34
  • Tm: 68°C, 71°C, 71°C


    • Thursday, 08/24/17

  • for results of test gel and proceeding please see low-copy lab book
  • Digestion and ligation of TAC3 taken from gel extraction (24.8., see low copy lab book) (also: digestion and ligation of PP7 -> discarded because futile)
  • Transformation of TAC3 into JM109 (Amp - backbone and Kan-Backbone)
  • for digestion see low copy lab book p.23
  • for ligation see low copy lab book p.24 (only tubes A3 and K3)


    • Tuesday, 08/29/17

  • Digestion of TAC2 taken from gel extraction (28.8., see low copy lab book)
  • At first not enough backbone (pSB1A3, pSB1K3, pSB1C3) for ligation -> please make sure to not accidentally put empty tubes back into the freezer
  • digested linearized backbones as done before (see high copy to do list p.3) to get more backbone for ligation also amplified linearized backbones (program: ampli Pauline in Torsten)
  • Ligation of TAC2 with the three newly digested backbones (low copy lab book p.24)
  • Transformation of all three ligations into JM109 (see transformation protocol, plated 200 µl)
    • -> Transformations in the 37° incubator
  • Colony-PCR of the TAC3-transformations from yesterday (Amp and Kan; see Colony-PCR protocol and note the changes in our folder!)
  • prepared 20 µl aliquots per clone, touched the six best looking clones with a tooth pick, tipped once on the masterplate and then stirred it in the aliquots
    • -> We had the problem that the toothpick absorbs the aliquot if left in it for too long
  • Gel picture looks good for both resistances, we inoculated two precultures per resistance for a miniprep tomorrow


    • Tuesday, 08/29/17

  • Not all TAC2 Transformations grew (TAC2+Kan missing) -> discarded (We kept the plates with TC2 + Amp and TAC2+CAP just in case)
  • Minipreps (see protocol, elution with 100 µl water)
    • TAC3 + KAN clone 2 231,3 ng/µl
    TAC3 + KAN clone 4 183,4 ng/µl
    TAC3 + AMP clone 4 314,5 ng/µl
    TAC3 + AMP clone 8 160,7 ng/µl
  • test digest of minipreps
  • Mastermix:
    • 5 µl CutSmart
    • 1 µl EcoRI-HF
    • 1 µl PstI
    • 33 µl water (total 40 µl, 8 µl per tube + two miniprep-plasmids)
  • gel picture see lab book, all clones positive
  • TAC3+Kan (clone 4) and TAC3+Amp(clone8) sent for sequencing
  • Therefor: 5 µl plasmid (100 ng(µl) + 5 µl primer (5 µM) Q 17
  • Glycerol stocks of TAC3+Kan (clone 4) and TAC3+Amp(clone8) -> 40% (1:1 dilution with 80% Glycerol)
  • prepared 3A assembly


    • Thursday, 08/31/17

  • diluted plasmids to 25 ng/µl (20 µl)
    • name c (ng/µl) plasmid (µl) water (µl)
    (C2) Cap +TAC1 265,7 1,9 18,1
    (A38) Amp + TAC1 189,5 2,6 17,4
    (K22) Kan +TAC1 241,4 2,1 17,9
    (C17) Cap + TAC3 293,6 1,7 18,3
    Klon 8, Amp +TAC3 160,7 3,1 16,9
    Klon 4, Kan + TAC3 183,4 2,7 17,3


    Friday, 09/01/17

  • Colony PCR with IG_C_K TaC1_2Fra and IG_C_K TaC3_2Fra (see protocol, wrong annealing Tm in To do list, use 52°C)
  • gel picture in lab book - no bands for TaC1 and wrong bands for TaC3 (approx 2200 bp)
  • next steps: rests of transformation (31.8.) of both replated on newly made plates (in case clone have been overgrown by other stuff because of not enough antibiotic in the Agar)
  • plated 50 µl and 200 µl
  • new ligation and transformation with rests of digest (31.8.) (in case transformation did not work)
  • To do: repeat colony PCR (in case that did not work)


    • Monday, 09/04/17

    observation of transformation plates:
  • new Trafo from 1.09 did not work, no growth on plates: Kan IG_C _K_ TaC1_2Fra Trafo 1.9 50µL and Kan IG_C_K_ TaC3_2Fra Trafo 1.9 50µL
  • transformation(31.8) new plated ones: colonies grew


    • Colony PCR of 4 plates called:
  • IG_C_K Tac1 _2 Fra Trafo31.08, plated 1.09 50µL
  • IG_C_K Tac1_2 Fra Trafo31.08, plated 1.09 200µL
  • IG_C_K Tac3_2 Fra Trafo31.08, plated 1.09 50µL
  • IG_C_K Tac3_2Fra Trafo31.08, plated 1.09 200µL
  • see colony pcr protocol, and 52 °C as annealing Tm, mastermix see lapbook
    • tubes 1-26:
  • 1-6 TaC1_2 50uL
  • 7-15 TaC1_2 200uL
  • 16-18 TaC3_2 50uL
  • 19-24 TaC3_2 200uL
  • tubes 25 and 26 were used as controls
  • 25 control well 18G (iGEM distribution kit Plate 4)
  • 26 control well 9D (IGEM distribution kit Plate 4)
  • run 1% agarose-gel at 120V for 1:15h
  • gel picture see lab book,
  • result: negative colony PCR ,no good bands ,were too small
    • -> better : use original plasmid psB1K3 which was already digested with E+P ,since this plasmid never contained a TaC
    -> assumption why colony PCR negative: the previously cut cut TaC from Kan TaC1 ligates with the plasmid hence the 3A assembly( Tac #(E+S) +TaC #(P+X) could not ligate in the vector
    -> better : ligation at 16°C for 2h or overnight


    restarted 3A assembly first cycle:
  • digestion: mastermix insert a, mastermix insert b see To do list table 16 page 15 used 4µL mastermix + 4µL plasmid(25 ng/µL)
  • tubes: for insert a: TaC1 C E+S; TaC3 C E+S for insert b: TaC1 A P+X; TaC3 A P+X


    • made 2ml overnight culture for glycerol stocks :
  • KAN TaC1 clone 4
  • Amp TaC1 clone 38
  • Cap TaC1 clone 2
  • KAN TaC3 clone 4
  • Amp TaC3 clone 4
  • KAN TaC3 clone 2
  • Cap TaC3 clone 17
  • Amp TaC3 clone8


    • Tuesday, 09/05/17

  • Ligation of digested TaCs from yesterday into pSB1K3 : according to To do list 3A assembly page 17
  • ligation reaction mix:

    		• TaC1_2Fra
    		 TaC3_2Fra
    T4 DNA Ligase 1µL 1µL
    T4 DNA Ligase buffer 1µL 1µL
    dwater 2µL 2 µL
    Cap TaC1 D E+S 2µL -
    A TaC1 D P+X 2 µL -
    Cap TaC3 D E+S - 2µL
    A TaC3 D P+X - 2µL
    vector pSB1K3 (digested, 29.08.) 2 µL 2 µL
  • in Torsten Thermocycler program 3A_Ligation
  • incubation at 16°C for 120 min
  • heat inactivation at 65°C for 10 min
  • hold at 10°C
    • -> rest of ligation reaction 5uL: in minitubes in the freezer (-20°C) in high copy box

  • Transformation of Ligation mix in JM109:
      • -used 5 µl of Ligation mix
      -used competent JM109 from promega
      -procedure according to transformation protocol
  • Glycerol stocks:
      • - for Kan TaC4 (Kl. 22), Amp TaC1 (Kl. 38), Cap TaC1 (Kl. 2), Kan TaC3 (Kl. 4), Amp TaC3 (Kl. 4), Kan TaC3 (Kl. 2), Cap TaC3 (Kl. 17), Amp TaC3 (Kl. 8)
      -mixed 80% glycerol 1:1 with liquid culture (700 µl + 700 µl)
      -stored in -80°C freezer, in dCas9 glycerol stock box


    Wednesday, 09/06/17

  • colony PCR of 3A Assembly
    • 12 clones from TAC1 and 12 clones from TAC3, with master plate
    • difficult to analyze (too many Nebenbanden)
    • expected sizes: TAC1 = 2022bp; TAC3 = 2232bp
    • we inoculate minipreps: tomorrow test digestion if the clones are positive or negative
  • minipreps of following clones (37° shaker)
    • TAC1: clone 5,6,7,8
    • TAC3: clone 5,6,7,10


    Thursday, 09/07/17

      *Miniprep of Tac1 (clone 5,6,7,8) anf TaC3 (clone 5,6,7,10)
      *elution in 50 uL ddwater
      *did glycerol stocks of TaC1 and TaC3 (0.3ml 80%Glycerol, 0,5ml TaC1/3)


    Friday, 09/08/17

  • Test digest of TaC1 (2 Fra) clone 5,6,7,8 and TaC3 (2 Fra) clone 5,6,7,10
    • used XbaI and PstI
    • gel picture: except for Tac1 clone 5 they all show correct bands
    • TaC1 clone 6 & 7 and TaC3 clone 5 & 6 prepared and sent for sequencing (5 µl DNA (100 ng/µl) + 5 µl Primer)
    • TaC1 clone 5 was discarded, TaC1 clone 8 and TaC3 clone 10 also ready for sequening, just in case something doesn't work with the sequencing from today




    Monday, 09/11/17

    3A assembly second cycle:
  • Master mix for insert b:
    • component volume [µl]
    10x NEBuffer 2.1 5
    PstI 0.5
    XbaI 0.5
    water 19
  • diluted TAC1 (two fragments) clone 8 and TAC3 (two fragments) clone 6 to 25 ng/µl
  • 4 µl of TAC1/3 (2frag) dilution + 4 µl Master mix (insert b)
  • incubation at 37°C for 1 h
  • insert a (TAC1/3 E+S) already digested (see 04.09.17)
  • vector pSB1C3 already digested (see 29.08.17)
    • Ligation of digested fragments:
    component volume used for TAC1 reaction [µl]
    		 volume used for TAC3 reaction [µl]
    water 1.4 1.2
    T4 DNA ligase buffer (10x) 1 1
    TAC1/3 (E+S) 2 2
    TAC1/3 (2frag, P+X) 2.6 2.8
    T4 DNA ligase 1 1
    sum 10 10
  • program for ligation in Torsten: 3A_ligation (16°C for 2 h, 65°C for 10 min)
  • Transformation of 5 µl of ligation reaction into competent JM109 from promega (see transformation protocol (plated on CAP plates: 200 µl + rest)
  • (rest of ligation reaction in -20°C freezer in high copy box)


    • Tuesday, 09/12/17

    Colony PCR (with Masterplate)
  • TAC1 (3 fragments, second cycle): 10 clones
  • TAC3 (3 fragments, second cycle): 10 clones
  • all clones negative


  • Gel Electrophoresis: 0,7%, 80V, 60min


    • Wednesday, 09/13/17

  • Minipreps from yesterday doesnt grow: new Minipreps inoculated (ca 8:30 am)
  • Miniprep (ca 16:15 pm)
  • 4ml per clone for miniprep, 1ml for Glycerolstock
  • TAC1: clone 2,4,6,7
  • TAC3: clone 1,2,4,8


    • Thursday, 09/14/17

    conzentration of the minipreps (13.09.)
    Tac 1 clone 2 37 ng/µl Tac 3 clone 1 134,6 ng/µl
    Tac 1 clone 4 45,2 ng/µl Tac 3 clone 2 80,7 ng/µl
    Tac 1 clone 6 70,1 ng/µl Tac 3 clone 4 94,0 ng/µl
    Tac 1 clone 7 32,4 ng/µl Tac 3 clone 8 94,9 ng/µl
    test digestion (60min at 37°C)
    Template [µl] Xba1 [µl] Pst1 [µl] ddH2O [µl] NEB 3,1 [µl]
    Tac 1 clone 2 5,4 0,5 0,5 2,6 1
    Tac 1 clone 4 4,5 0,5 0,5 3,5 1
    Tac 1 clone 6 2,9 0,5 0,5 5,1 1
    Tac 1 clone 7 6,2 0,5 0,5 1,8 1
    Tac 3 clone 1 1,5 0,5 0,5 6,5 1
    Tac 3 clone 2 2,5 0,5 0,5 5,5 1
    Tac 3 clone 4 2,2 0,5 0,5 5,8 1
    Tac 3 clone 8 2,1 0,5 0,5 5,7 1
  • gel electrophoresis
    • 0,7% gel
    • 30min at 120V



  • Glycerolstocks (700ul Template + 700ul 80% Glycerol)
    • Tac1 clone 4 IG_C_TH13
    Tac3 clone 1 IG_C_TH15 cl1
    Tac3 clone 8 IG_C_TH15 cl 8
    5) 3A Assembly , third cycle (HighCopy ToDo page 27)
    with: Tac1 clone 4 and Tac3 clone 8
    5a) digestion of templates
    component Mastermix insert (a) Mastermix insert (b) Mastermix vector
    10x NEB 2 Buffer 5 µL 5 µL 5 µL
    EcoRI-HF 0.5 µL --- 0.5 µL
    PstI --- 0.5 µL 0.5 µL
    SpeI 0.5 µL --- ---
    XbaI --- 0.5 µL ---
    dH2O 19 µL 19 µL 19 µL
    Preparation of the Mastermix for the different templates in cycle 3
    name V(mastermix) insert (a) V(mastermix) insert (b) V(mastermix) vector Used Plasmid backbone (25 ng/µL)
    IG_C_K_TaC_1_D_E+S 4 µL --- --- IG_C_K_TaC_1 (4µL)
    IG_C_K_TaC_3_ D_E+S 4 µL --- --- IG_C_K_TaC_3 (4µL)
    IG_C_ CAP_TaC_1_3Fra_D_P+X --- 4 µL --- IG_C_ CAP_TaC_1_3Fra (4µL)
    IG_C_ CAP_TaC_3_3Fra _ D_P+X --- 4 µL --- IG_C_ CAP_TaC_3_3Fra (4µL)
    IG_C_ A_TaC_1_ D_E+P --- 8 µL IG_C_A_TaC_1 (8µL)
    Digestion reaction for the inserts (a) and (b) for each target cassette type in cycle 3
  • mix components by pipetting up and down or "flicking" the tube.
  • microfuge briefly, Don’t vortex!
  • incubate for 30 min at 37 °C
  • heat kill for 20 min at 80 °C
  • store at -20 °C


    • 5b) ligation of target cassettes and plasmid backbones
    The ligation is done by using the T4 ligase. In the end, three different ligation reactions should be prepared, so that each plasmid consists of four target cassettes of its corresponding type. The solutions containing the digested backbones from 5a) are used. The inserts are used an equimolar amount. In a reaction volume of 20 µL, 0.02 pmol of each template are used.
    Component Target cassette type 1 IG_C_A_TaC_1_4Fra Target cassette type 3 IG_C_A_TaC_3_4Fra
    T4 DNA Ligase 1 µL 1 µL
    T4 DNA Ligase Buffer 0.5 µL 0.5 µL
    dH2O 2.5 µL 2.5 µL
    IG_C_K_TaC_1_D_E+S 2 µL ---
    IG_C_K_TaC_3_ D_E+S --- 2 µL
    IG_C_ CAP_TaC_1_3Fra_D_P+X 3.2 µL ---
    IG_C_ CAP_TaC_3_3Fra _ D_P+X --- 3.5 µL
    IG_C_ A_TaC_1_ D_E+P 2 µL 2 µL
    Preparation of the ligation reaction
  • gently mix the reaction by pipetting up and down and microfuge briefly
  • incubation at 16 °C overnight


    • Friday, 09/15/17

  • heat inactivation at 65°C for 10 min
  • chill on ice
    • 5c) Transformation in chemical component JM109 cells
  • thaw tubes of chemical competent JM109 cells on ice for 10 min
  • add 50 µL of competent cells to 2 µL of each ligation reaction
  • chill on ice for 30 min
  • heat shock for 30 sec
  • chill on ice for 5 min
  • add 950 µL SOC media (room temperature) to transformation tubes
  • incubate at 37 °C for 60 min (shake 850 rpm)
  • plate sample on the different LB-agar plates (Ampicillin resistance)
    • 200 µL of each transformation
    • also: centrifuge the rest, discard the flowthrough (leave just one drop and resuspend with them the cells) and put this on the plate
  • incubation till 18.09.2017 at room temperature


    • Monday, 09/18/17

  • made master plates (amp) for TAC1_4Fra and TAC3_4Fra from trafo plates (15.09.)
    • picked 18 clones per TAC
    • only made this plate to secure clones
  • inoculation of liquid culteres for clon 1-6 (for each TAC) -> in total 12 minipreps
    • for minipreps tomorrow
    • 5 ml LB + amp


    Tuesday, 09/19/17

    Clean up of minipreps from yesterday (TaC1/3_4 Fra)
    • elution in 50 µl water

    Test digest with mastermix for 12 minipreps + 2 as buffer:

    NEBuffer 14 ml
    PstI 2,8 ml
    XbaI 2,8 ml
    water 92,4 ml
    mastermix composition

  • step by step:
    • 8 µl master mix + 2 µl miniprep per aliquot
    • incubation for 2h at 37°C
    • test gel: 10 µl digest + 2 µl loading Dye
    • 0,7% agarose; 120V for 45 min + 140V for 10 min
    • gel picture: TaC1/3 with 4 fragments
    • TaC1 clone 1 and 6 positive
    • TaC3 clone 2 positive
    • expected lenghts: Backbone 2000 bp, TaC1_4Fra ~3500 bp, TaC3_4Fra ~3900 bp
    • prepared glycerol stocks (1:1 with 86% glycerol)
    • Names: IG_C_A_TaC1_4 Fra & IG_C_A_TaC3_4 Fra


    Wednesday, 09/20/17

    3A Assembly – fourth assembly cycle
    In this first assembly cycle two Inserts are combined, so that the product plasmid contains five target cassettes. The inserts (a) are digested using EcoRI and SpeI, the inserts (b) using XbaI and PstI. The digestion an ligation is done following the “3A Assembly”protocol of iGEM.
    Digest
    component Mastermix insert (a) Mastermix insert (b)
    10x NEB 2 Buffer 5 µL 5 µL
    EcoRI-HF 0.5 µL ---
    PstI --- 0.5 µL
    SpeI 0.5 µL ---
    XbaI --- 0.5 µL
    dH2O 19 µL 19 µL
    Preparation of the Mastermix for the different templates in cycle 4
    name Vmastermix insert (a) Vmastermix insert (b) Used Plasmid backbone (25 ng/µL)
    IG_C_ CAP_TaC_1_D_E+S 4 µL --- IG_C_ CAP_TaC_1 (4µL)
    IG_C_ CAP_TaC_3_ D_E+S 4 µL --- IG_C_ CAP_TaC_3 (4µL)
    IG_C_ A_TaC_1_4Fra D_P+X --- 4 µL IG_C_ A_TaC_1_4Fra (4µL)
    IG_C_ A_TaC_3_4Fra D_P+X --- 4 µL IG_C_ A_TaC_3_4Fra (4µL)
    Digest reaction for the inserts (a) and (b) for each target cassette type in cycle 4
  • mixing components by "flicking" the tube
  • microfuge briefly
  • incubation for 30 min at 37 °C
  • heat kill for 20 min at 80 °C
    • Ligation
    Component Target cassette type 1 IG_C_K_TaC_1_5Fra Target cassette type 3 IG_C_K_TaC_3_5Fra
    T4 DNA Ligase 1 µL 1 µL
    T4 DNA Ligase Buffer 0.5 µL 0.5 µL
    dH2O 2.5 µL 2.5 µL
    IG_C_ CAP_TaC_1_D_E+S 2 µL ---
    IG_C_ CAP_TaC_3_ D_E+S --- 2 µL
    IG_C_ A_TaC_1_4Fra D_P+X 3.8 µL ---
    IG_C_ A_TaC_3_4Fra D_P+X --- 4.1 µL
    IG_C_ K_TaC_1_ D_E+P 2 µL 2 µL
    Preparation of the ligation reaction
  • gently mixing the reaction by pipetting up and down and microfuge briefly
  • Incubation at 20 °C for 150 min
  • Heat inactivation at 65°C for 10 min
  • Transformation with chemical competent JM109 cells


    • Thursday, 09/21/17

  • inoculate preculture Miniprep (incubate 37° over night with shaking)
    • Clone TaC1 V Media, Antibiotica Clone TaC3 V Media, Antibiotica
    clone 1 5ml, KAN clone 5 5ml, KAN
    clone 2 5ml, KAN clone 6 5ml, KAN
    clone 3 5ml, KAN clone 7 5ml, KAN
    clone 4 5ml, KAN clone 8 5ml, KAN


    Friday, 09/22/17

    miniprep of Tac1 (clone 1,2,3,4) anf TaC3 (clone 5,6,7,8)
    concentration in ng/µl
    		concentration
    Tac 1 clone 1 585,9 Tac 3 clone 5 527,7
    Tac 1 clone 2 668,0 Tac 3 clone 6 535,8
    Tac 1 clone 3 584,3 Tac 3 clone 7 450,1
    Tac 1 clone 4 533,6 Tac 3 clone 8 563,1
    Test digest with mastermix for 8 minipreps + 1
    NEBuffer 2.1 9 µl
    PstI 1,8 µl
    EcoRI 1,8 µl
    water 68,4 µl
  • 9 µl master mix + 1 µl miniprep per aliquot
  • incubation for 1h at 37°C
  • test gel: 10 µl digest + 2 µl loading Dye
  • 0,7% agarose; 120V for min
    • ->result: TaC1 clone 4 positive
  • TaC3 clone 8 positive
  • glycerol stocks (1:1 with 86 % glycerol)
  • names: TAC1: iG_C_TH19 und TAC3:IG_C_TH21