Tianjin/FLIP-FLOP/More details

From 2007.igem.org

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<td><font size="3" color="#9966CC">10μL digesting Mix</font>
<td><font size="3" color="#9966CC">10μL digesting Mix</font>
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<li>1.0 μL 10X Enzyme buffer </li>
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<li>0.5 μL Restriction Enzyme respectively</li>
<li>0.5 μL Restriction Enzyme respectively</li>
<li>Add ddH<sub>2</sub>O to 10ul volume</li>
<li>Add ddH<sub>2</sub>O to 10ul volume</li>
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<td><font size="3" color="#9966CC">50μL digesting Mix</font>
<td><font size="3" color="#9966CC">50μL digesting Mix</font>
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<li>5.0 μL 10X Enzyme buffer </li>
<li>5.0 μL 10X Enzyme buffer </li>
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<li>2 μL Restriction Enzyme respectively</li>
<li>2 μL Restriction Enzyme respectively</li>
<li>Add ddH<sub>2</sub>O to 50 ul volume</li>
<li>Add ddH<sub>2</sub>O to 50 ul volume</li>
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Revision as of 18:36, 26 October 2007

Experiment

  • E.coli Cultivate

    • Solid LB culture is used for agar plate. For a typical liquid culture, use 5 ml or 100ml of appropriate medium, and condition is supposed to be 37°C and 220rpm. The amount of time you wait depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, wait about 12-16 hours.

  • Plasmid Extraction

    • Using plasmid extracting kit we get the plasmid DNA for two purposes. One is verifying the plasmid using enzyme digesting and the other is purifying the fragment using enzyme digesting and agarose gel electrophoresis.

  • Verification

    • Colony PCR can be used after a transformation to screen colonies for the desired plasmid.But it always give the incorrect result, so often 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 gotten 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 is decided by formula given by openwetware.

    Reaction
    Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total volume is 10μL, one half is DNA solution and one half is Solution I. Temperature is 16°C. Then waiting for different time according to different situations.

  • E.coli Transformation

    • Using CaCl2 solution to make competent cells, and chemically transform foreign DNA 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 jproduct to 100 μL competent cells.

  • Detection

    • Open the bottle of medium and flame the mouth, measure out 2 ml to fill 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 fluorometry. AHL is detected by the detector used in Bio-Diode.

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