Team:Chiba/Notebook/protocol

From 2009.igem.org

E.coli Time Manager


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The Project

1, Introduction

2, Project Design

3, Experiments, Results & Discussion

3-1, Making LuxR Mutants

3-2, Characterization of LuxR Mutants

3-3, Demonstration

5, Conclusions

Contents

  1. Transformation (Using Zymo comp.)
  2. DNA_Purification
    1. Sigma prep (Plasmid mini prep)
    2. Zymo DNA Cleam&Concentrator Kit
  3. Agarose gel electrophoresis
  4. Digestion
  5. PCR
  6. Gel extract
  7. Dephosphorylation of DNA
  8. Ligation

Protocols

Transformation

Day 1 morning

  • 100 ml SOB medium in 1L or 500 mL flask and sterilize
  • E.coli culture grown in 2 mL of fresh LB medium.

Day 1 night

  • Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 °C until OD is 0.4-0.6.

Day 2

  • Transfer the culture to ice 10min.
  • Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)
  • Pellet the cells by centrifugarion at 2500rpm for 6 min.
  • Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).
  • Pellet the cells by centrifugarion at 2500rpm for 6 min.
  • Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).
  • Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.


DNA Purification

Sigma prep

Zymo DNA Cleam&Concentrator Kit

  • Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.
  • Load mixture silica column and place column into a 2 ml collection tube
  • Centrifuge at full speed for 30 sec. Discard the flow-through.
  • Add 200μL of wash buffer and spin 30 sec.
  • Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA.


Agarose gel electrophoresis

  • Agalose Gel casting
  1. Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer
  2. Microwave until the agarose is fully melted
  3. Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid
  4. Remove comb


  • Running agalose gel
  1. Load 5 μL prepared 1kbp ladder
  2. Mix DNA solution with loading dye(6x) and water
  3. Load it into agalose gel
  4. Run the gel at ~100 volts for 35 mins.


  • Visualizing agarose gels
  1. Remove gel from gel box
  2. Soak the gel in ethidium bromide solution
  3. Let it 30 min.
  4. Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
  5. Print the picture.
  6. Remove gel and throw in trash
  7. Wipe down Trans-Illuminator if necessary.


Digestion

  1. Mix (in a PCR tube)
    1. plasmid DNA
    2. buffer
    3. Restriction Enzyme
    4. NFW
  2. Incubate at 37ºC for 3h

PCR

  • Resuspend primer in Nuculease free water to 100 µM
  • PCR mix
  1. DNA template 1µL
  2. Fwd primer 10µL (final con. 10 pM)
  3. Rev primer 10µL
  4. 10x thermo pol buffer 10µL
  5. dNTP mix 10µL
  6. DNA pol. 1µL
  7. dH20 58µL


----------------------------
100µL


  • PCR cycle 
Start: 94 °C for 5 min. (melt)
cycle:          melt:            1 min. 
             anneal :           30 sec. 
cycle end: extension:72 °C for 3.5 min. 
25 cycles 
72 °C for 10 min
store: keep at 6 °C forever


Gel extract

  • Agalose Gel casting
  1. Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer
  2. Microwave until the agarose is fully melted
  3. Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid
  4. Remove comb


  • Running agalose gel
  1. Load 5 μL prepared 1kbp ladder
  2. Mix DNA solution with loading dye(6x) and water
  3. Load it into agalose gel
  4. Run the gel at ~100 volts for 35 mins.


  • Visualizing agarose gels
  1. Remove gel from gel box
  2. Soak the gel in ethidium bromide solution
  3. Let it 30 min.
  4. Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
  5. Print the picture.
  6. Remove gel and throw in trash
  7. Wipe down Trans-Illuminator if necessary.


  • Extract
  1. Cut the agarose target band
  2. The chip of the gel into 2mL ADB buffer
  3. Let it in 37 degree 30 min to solve the agarose gel.
  4. Purify the DNA with Zymo DNA Cleam&Concentrator Kit

Dephosphorylation of DNA

SAP: Alkaline Phosphatase (Shrimp)

  1. Mix
    1. DNA fragment 1~10 pmol
    2. shrimp Alkaline Phosphatase (1~5 μ l) 1~5 U
    3. 10X SAP Buffer 5 μ l
    4. Sterilized distilled water up to 50 μ l
  2. Incubate at 37°C for 15~30 min.
  3. Incubate at 65°C for 15 min. (for inactivation by heat treatment)
  4. Purify the DNA with Zymo DNA Cleam&Concentrator Kit


Ligation

  1. Mix insert DNA with vector DNA.
  2. Add 1u Invitrogen Ligase and Ligase-Buffer(x5).
  3. Store RT for 3h.

Time Delay Test

  1. Transformed sender and receiver into E coli strains.
  2. Inoculated them independently in liquid media. Incubated at 37°C 12h.
  3. Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
  4. Washed sender and receiver.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 25°C, 30°C　or 37°C.
  7. Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR　Thermo ELECTRON CORPORATION)
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