Tianjin/FLIP-FLOP/More details

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Experiment

  • E.coli Cultivation

    • Solid LB medium is used for agar plate. For a typical cultivation, 5 ml or 100ml of appropriate medium is used, and the appopriate condition for cultivation in shakers is 37°C and 220rpm and the time depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, waiting for about 12-16 hours.

  • Plasmid Extraction

    • Using plasmid extracting kit we get the plasmid DNA for two purposes. One is to verify the plasmid using enzyme digesting and the other is to purify the fragments for ligation using enzyme digesting and gel recycle.

  • Verification

    • Colony PCR can be used to screen colonies containing desired plasmid.But it always brings out the incorrect result, so under most conditions we use enzyme digesting to verify the desired palsmid.

  • Restrication digestion
    • 10μL digesting Mix
    • 1.0 μL 10X Enzyme buffer
    • 5 μL plasmid DNA
    • 0.5 μL Restriction Enzyme respectively
    • Add ddH2O to 10ul volume
    		 50μL digesting Mix
    • 5.0 μL 10X Enzyme buffer
    • 30 μL plasmid DNA
    • 2 μL Restriction Enzyme respectively
    • Add ddH2O to 50 ul volume
  • Agarose gel electrophoresis
    • Agarose concentration : 1.0g/100mL
    • Buffers: TAE - better resolution of fragments >4kb
    • The appropriate volume: 30 ml for fragment verifying or 50 ml for fragment purification
    • Voltage: 80-100V
  • DNA ligation

    • The fragment usually is prepared from gel purification, sometimes from nucleic acid co-depositing.
    Reagents

    • T4 DNA ligase
    • 10x T4 DNA Ligase Buffer
    • Deionized, sterile H2O
    • Purified, linearized vector in EB
    • Purified, linearized insert in EB

    Calculating
    The amount of vector and insert fragment needed for a successful ligation is decided by formula given by openwetware.

    Reaction
    Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total reaction volume is 10μL, one half of which is DNA solution and another half is Solution I containing T4 ligation enzyme. Typical reaction temperature is 16°C. Reaction time varies from 45 minutes to 6 hours depending on the specific condition.

  • E.coli Transformation

    • Using CaCl2 solution to make competent cells, and foreign DNA is transformed chemically to competent cells. If foreign DNA is plasmid, using 2-3 μL to transform 100 μL competent cells. If it is ligation product, transform the total 10 μL product to 100 μL competent cells.

  • Detection

    • Opening the bottle of medium and flame the mouth, bringing 2 ml to 10 ml Eppendorf Tube.Centrifugate the tube and pipette in 5 ml of the suspension to another tube for detecting the extracellular AHL.Then the deposit is used to detect GFP using fluorometer. The concentration of AHL is detected by the detector used in Bio-Diode.

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