Team:UNESP Brazil/Notebook

Notebook

  • Test-tube culture for PCR (tetR and SpdCas9)

    Test-tube 405 (Pveg-cre-tetR) - Chloramphenicol

    Test-tube 472 - Ampicillin

        

    Plasmid Miniprep (405 and 472)

    Pveg-cre-tetR: 100,1 ng/uL

    SpdCas9: 37,3 ng/uL

    February 2021

  • PCR (SpdCas9 and tetR)

    SpdCas9: 4327 bp - Tm: 65°C - Extension time: 2:30 min

    tetR: 796 bp - Tm: 60 °C - Extension time: 1 min

    Template - 50ng

        

    Digestion (SpdCas9,TetR, pSB1A3 - 37°C - 30 min)

    pSB1A3: 100 ng - EcoRI + PstI + FastAP

    tetR: 1000 ng - EcoRI + BcuI

    SpdCas9: 1000 ng - XbaI + PstI

        

    Ligation (22°C - 1h)

    Ligation calculator - 1:3

    pSB1A3(25 ng) + tetR(27,7 ng) + SpdCas9(150,5 ng)

        

    Transformation

    50 uL E. coli + 10-50 ng DNA (~10 uL)

    Heat-shock: ice (30 min) + 42°C (30 sec) + ice (2 min)

    Incubation with SOC: 37°C - 1 hour

        

    Test-tube culture (six colonies)

    Digestion (EcoRI + PstI and EcoRI + SpeI and EcoRI + XhoI)

    March 2021

  • gBlocks PCR

    BBprefix_fw + BBsuffix_rv + Taq DNA pol + 50 ng DNA template

        

    E. coli Top10 chemically competent

    Follow protocol - dropbox

        

    Digestion (pSB1A3, pSB1K3, 9.1, 9.2)

    pSB1A3: 500 ng - XbaI + PstI + FastAP

    pSB1K3: 500 ng - XbaI + PstI + FastAP

    9.1: 500 ng - XbaI + PstI

    9.2: 500 ng - XbaI + PstI

    Ligation (22°C - 1h)

    Ligation calculator - 1:3

    pSB1K3(25 ng) + 9.1(9,75 ng)

    pSB1K3(25 ng) + 9.2(12,5 ng)

        

    Transformation pSB1K3 (iGEM Kit)

    Miniprep pSB1K3 + Criotube

        

    Digestion (10.1, 10.2)

    10.1: 500 ng - XbaI + PstI

    10.2: 500 ng - XbaI + PstI

    pSB1A3: 1000 ng - XbaI + PstI + FastAP

    Ligation + Transformation

        

    Digestion pSB1K3

    Overnight - too many red colonies

    Transformation 10.1/10.2 + pSB1K3

        

    Test-tube 10.1, 10.2

    Digestion to confirm cloning

    AsiA PCR to correct reading frame

    2-3 ng DNA template

    Digestion pSB1A3_tetR_SpdCas9

    Digestion AsiA

    AsiA: 500 ng - BcuI + PstI - overnight

    August 2021

  • Ligation pSB1K3 + 9.1/9.2/10.1/10.2

        

    Criotube 9.1/9.2

        

    Digestion to confirm cloning 9.1/9.2/AsiA

    XbaI + PstI

        

    Sequencing AsiA - primer 618 - Tm 60°C

    Digestions 9.1, 9.2, 10.1, 10,2 and ligation with 11

        

    Test-tube pSB1K3_(9.1/9.2/10.1/10.2)_11 - SUCCESS WITH 9.1 AND 9.2

    Golden Gate 8.1+8.2

    PCR after Golden Gate - Tm 58°C - primers 123+124 (BBprefix and BBsuffix)

    September 2021

  • Repeat AsiA PCR

    Ta 55°C - Time-Hifi - primers 124+635 - SUCCESS

    PCR target_broccoli

    Ta 55°C - Time-Hifi - primers 123+124

        

    Digestion pSB1A3_tetR_SpdCas9, AsiA, target-broccoli, 11

    pSB1A3_tetR_SpdCas9: 500 ng - BcuI + PstI + FastAP - 3 hours - 37°C

    AsiA: 500 ng - PstI + XbaI - 3 hours - 37°C

    target-brococli: 500 ng - EcoRI + BcuI - 1 hour - 37°C

    11: 500 ng - EcoRI + BcuI - 1 hour - 37°C

        

    Ligation

    9.1_11/9.2_11 + target-broccoli

    10.1/10.2 + 11 - SUCCESS - DIGESTION ELECTROPHORESIS

    pSB1A3_tetR_SpdCas9 + AsiA

    Negative control

        

    Repeat AsiA ligation

    1:3, 1:6, 1:10, 1:50

    AsiA: 5 ng

    plasmid: 25 ng

    Transformation AsiA + Incubation (overnight, 37°C)

    Repeat PCR target-broccoli

    Criotube pSB1A3_tetR_SpdCas9_AsiA

    Digestion to confirm Asia: colonies 1, 2 and 9 - PCR

    PCR to confirm: primers 635/124 - Tm 60°C

    Colony (1) - OK

    Transformation 9.1/9.2 + target-broccoli

    Digestion to confirm target-broccoli: E + P - SUCCESS

    Transformation BL21

    1. pSB1A3_tetR_SpdCas9_AsiA + 9.1_8/9.2_8/10.1_11/10.2_11 (4 plates Kanamycin+Chloramphenicol+Ampicilin)

    2. pSB1A3_tetR_SpdCas9_AsiA (1 plate Chloramphenicol+Ampicilin)

    3. 9.1_8/9.2_8/10.1_11/10.2_11 (4 plates Kanamycin+Chloramphenicol)

    October 2021