Team:Tsinghua/Protocol

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Protocols

1. Plasmid DNA purification
(1)Centrifuge the 5mL culture tube for 10 minutes at 3000 rpm.
(2)Remove the culture medium and add 250uL buffer P1 (RNase added and 4°C stored) to resuspend the pelleted bacteria.
(3)Transfer to a 1.5mL EP tube.
(4)Add 250uL buffer P2 and gently mix by inverting the tube 8 times.
(5)Add 350uL buffer P3 and immediately mix by inverting the tube 8 times.
(6)Centrifuge the tube for 10 minutes at 12000 rpm.
(7)Transfer the supernatant to a pre-balanced (by buffer BL) column.
(8)Centrifuge the column for 60 seconds at 12000 rpm.
(9)Remove the flow-through and add 600uL buffer PW (alcohol added).
(10)Centrifuge the column for 60 seconds at 12000 rpm.
(11)Repeat step 9-10.
(12)Remove the flow-through and centrifuge for another time.
(13)Place the column in a 1.5mL EP tube and wait for 2 minutes at RT.
(14)Add 60uL ddH2O onto the column and centrifuge for 60 seconds at 12000 rpm.
(15)Measure the concentration of plasmid by Nanodrop.

2. Gel extraction
(1)Remove the gel containing DNA of interest and place it into a 1.5mL EP tube.
(2)Add 500uL solution SN per 100mg agarose gel and dissolve the gel for 5 minutes at 65°C.
(3)Add 50uL solution B per 500uL solution SN and mix them up.
(4)Place the 3S column into the collecting tube and transfer the solution mixture into the column.
(5)Place the column for 2 minutes at RT and then centrifuge for 1 minute at 10000 rpm.
(6)Discard the flow-through and add 600uL Wash solution.
(7)Centrifuge for 1 minute at 10000 rpm.
(8)Repeat step 6-7.
(9)Place the column into a new 1.5mL EP tube and add 30uL ddH2O.
(10)Place the column for 2 minutes at RT and centrifuge for 1 minute at 10000 rpm.
(11)Measure the concentration of DNA by Nanodrop.

3. Chemical transformation
(1)Add 5uL plasmid into 50uL competent DH5α and place the tube on ice for 30 minutes.
(2)Heat up the tube for 90 seconds at 42°C.
(3)Place the tube on ice for 2.5 minutes.
(4)Add 1mL LB (without antibiotics) on a germ-free bench.
(5)Recover the bacteria by shaking the tube for 30 minutes at 37°C.
(6)Centrifuge (if needed) for 3 minutes at 5000 rpm and remove most of the supernatant.
(7)Resuspend the bacteria and add 100uL suspension onto the plate with antibiotics.
(8)Culture overnight at 37°C.

4. Transformation protocol for distribution kit
(1)Insert a tip into the well containing plasmid of interest.
(2)Add 10uL ddH2O and dissolve the plasmid for 5 minutes.
(3) 5uL for transformation.

5. Electro-transformation
(1)Produce electroporation-competent cells.
(2)Add 50uL bacteria into 100mL LB and culture at 30°C until its optical density (OD600) reaches 0.05-0.1. Then add 1mM L-arabinose until it reaches 0.6. Place the cells on ice for 1 hour and then wash with overnight-stored cold H2O twice and cold 10% glycerol once. Condense 100 times and dispense 50uL into each tube. Store at -80°C.
(3)Electro-transform 10-100ng DNA per 50uL cells using Eporator from Eppendorf with 1mm3 electric shock cup (Electric shock is given at 1.8kV for 4-5 seconds).
(4)Recover the bacteria in 1mL LB at 37°C.
(5)Plate the bacteria on the plate with antibiotics.

6. PCR
(1)Commonly used system:

  • KOD buffer: 1uL
  • dNTP: 1uL
  • MgSO4: 1uL
  • Forward primer: 0.2uL
  • Reverse primer: 0.2uL
  • KOD plus: 0.2uL
  • Template: 0.2uL (it depends)
  • ddH2O: add up to 10uL
    Total volume: 10uL

    (2)Commonly set up program:

  • 94°C 5 minutes
  • 94°C 30 seconds
  • 60°C (it depends) 30 seconds    30 cycles (it depends) 
  • 72°C 30 seconds(it depends)
  • 72°C 7 minutes
  • 12°C ∞

    7. RNA extraction
    (1)Homogenize the tissue by grinding the tissue in liquid nitrogen with motar and pestle.
    (2)Transfer the mixture into a tube and add TRIzol to stabilize the RNA.
    (3)Incubate the sample for 5 minutes at RT.
    (4)Add chloroform (1:5 volume ratio of chloroform to TRIzol is preferred) to the sample.
    (5)Mix the sample by inverting for 8 times.
    (6)Incubate for 5 minutes at RT.
    (7)Centrifuge at 12000 g for 5 minutes at 2-8°C.
    (8)Transfer the colorless aqueous upper layer to a new tube.
    (9)Add isopropanol (1:2 volume ratio of isopropanol to TRIzol is preferred) to the sample.
    (10)Mix the sample by inverting for 8 times.
    (11)Incubate for 5 minutes at RT.
    (12)Centrifuge at 12000 g for 10 minutes at 4°C.
    (13)Remove supernatant and add 70% ethanol.
    (14)Mix by vortexing shortly.
    (15)Centrifuge at 12000 g for 10 minutes at 4°C.
    (16)Air-dry the pellet for 5 minutes.
    (17)Dissolve the pellet with 50uL DEPC H2O.
    (18)Mix the solution by inverting for several times and heat for 10 minutes at 55°C.
    (19)Measure the concentration of RNA by Nanodrop.

    8. Reverse transcription PCR
    (1)Template RNA is incubated with primer pairs at 70°C to denature secondary structures.
    (2)Quickly place the mixture on ice.
    (3)dNTP, RNase inhibitors, reverse transcriptase and reaction are then added.
    (4)Incubate the mixture for 1 hour at 42°C.
    (5)Place the mixture at 70°C to inactivate the enzyme.
    (6)Use the newly synthesized DNA as the template for future experiments.

    9. Enzymatic digestion

  • Buffer: 1uL
  • Template: 2uL (it depends)
  • Restriction enzyme: 0.2-0.5uL
  • ddH2O: add up to 10uL
    Total volume: 10uL
    Place at 37°C for 1 hour

    10. Ligation

  • 10×T4 ligase buffer (NEB) 1uL
  • 100×BSA 0.1uL
  • T4 ligase 0.2uL
  • Linearized backbone 0.1uL (it depends)
  • Insert 1uL (it depends)
  • BsaI 0.5uL
  • ddH2O add up to 10uL
    Total volume: 10uL
    Notes: The molecular weight ratio of insert to backbone that is used commonly is 3:1.

    11. Seamless cloning
    (1)Add homologous overlap (usually 15-30 bp) to DNA fragments.
    (2)Purify the DNA fragments and measure its concentration.
    (3)Set up the assembly cloning reaction with the following system:

  • DNA fragments + linearized vector 0.5-5uL
  • 2× Reagent mix 5uL
  • ddH2O add up to 10uL
    Total volume: 10uL

    (4)Place the reaction mix for 15 minutes at 50°C.
    (5)Use 5uL mix for transformation.
    Note: Ratio between inserts and ratio between vector and inserts should be kept at 1:1 and 1:3 respectively.

    12. Bacterial genome one-step knock-out assay
    Adapted from method designed by Wanner.
    Prepare the following plasmids:

     

    Requirement from the host bacteria

    Resistance gene

    Temperature requirement

    pKD46

     

    Amp

    30°C

    pKD3/4/13

    BW25141

    Amp, Kan or Cm

     

    pCP20

     

    Amp, Cm

    30°C

    1. Transform pKD46 (temperature-sensitive, culture at 30°C) and screen on Amp+ plate.
    2. Extract the plasmid and confirm.
    3. Amplify the Kanamycin gene to be inserted with primers share homology (usually 39bp for homologous arm and 20bp for matching sequence of Kan) to the genome by PCR.
    4. Electro-transform the fragment after PCR and screen on Kan+ plate.
    5. Pick multiple colonies and preserve on Kan+ plate.
    6. Meanwhile confirm the colonies by colony PCR.
    7. Culture in LB at 37°C without antibiotics overnight in order to remove pKD46.
    8. Culture on plate at 30°C without antibiotics.
    9. Pick colony and screen positively using Kan+ plate.

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