Difference between revisions of "Team:IISc-Bangalore/Protocols"

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<div id="inPageNav">
 
<div id="inPageNav">
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            <ol>
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<ol>
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                <li>Transformation</li>
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<li>Transformation</li>
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                <li>Plasmid isolation</li>
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<li>Plasmid isolation</li>
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                <li>PCR</li>
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<li>PCR</li>
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                <li>DNA purification</li>
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<li>DNA purification</li>
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                <li>Restriction digestion</li>
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<li>Restriction digestion</li>
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                <li>Ligation</li>
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<li>Ligation</li>
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                <li>SEM assay</li>
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<li>SEM assay</li>
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                <li>DLS assay</li>
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<li>DLS assay</li>
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                <li>Spectrophotometry assay</li>
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<li>Spectrophotometry assay</li>
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                <li>Reagents</li>
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<li>Reagents</li>
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            </ol>
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</ol>
 
</div>
 
</div>
  
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     <h2>Preparation of competent cells - TSS method</h2>
 
     <h2>Preparation of competent cells - TSS method</h2>
 
         <h3>Day 1</h3>
 
         <h3>Day 1</h3>
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<ol>
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<table style="width:100%">
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    <li>Prepare a primary inoculum of <i>E. coli</i> DH5α in 5 mL LB in a test tube.</li>
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<tr>
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    <li>Incubate overnight at 37°C, 170 rpm.</li>
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<th>Step</th>
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</ol>
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<th>Description</th>
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<h3>Day 2</h3>
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<th>Rationale</th>
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</tr>
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<tr>
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<td>1</td>
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<td>Prepare a primary inoculum of <i>E. coli</i> DH5α in 5 mL LB in a test tube.</td>
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<td>-</td>
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</tr>
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<tr>
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<td>2</td>
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<td>Incubate overnight at 37°C, 170 rpm.</td>
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<td>-</td>
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</tr>
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</table>
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<h3>Day 2</h3>
 
<table style="width:100%">
 
<table style="width:100%">
 
<tr>
 
<tr>

Revision as of 12:20, 22 October 2017

  1. Transformation
  2. Plasmid isolation
  3. PCR
  4. DNA purification
  5. Restriction digestion
  6. Ligation
  7. SEM assay
  8. DLS assay
  9. Spectrophotometry assay
  10. Reagents

Transformation

Preparation of competent cells - TSS method

Day 1

Step Description Rationale
1 Prepare a primary inoculum of E. coli DH5α in 5 mL LB in a test tube. -
2 Incubate overnight at 37°C, 170 rpm. -

Day 2

Step Description Rationale
1 Inoculate 1 mL overnight culture in 100 mL LB medium (1% inoculum) -
2 Incubate at 37°C, 170 rpm until the OD reaches 0.4 (~1 h 45 min for E. coli DH5α cells) -
3 Place the culture at 4°C for 45 min -
4 Spin down the culture at 10000 rpm, 10 min, 4°C -
5 Resuspend the cell pellet in 1 mL ice-cold TSS buffer -
6 Spin down the culture at 10000 rpm, 10 min, 4°C -
7 Make 50-100 uL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage -

Heat-shock transformation

Plasmid isolation

Miniprep

Midiprep

PCR

Simple PCR

Overhang PCR

Colony PCR

DNA purification

Chloroform extraction

Gel purification

Restriction digestion

Single digestion

Double digestion

Overnight digestion

Ligation

Simple ligation

Sequential ligation

Multi-ligation

Scanning Electron Microscopy (SEM) assay

Dynamic Light Scattering (DLS) assay

Spectrophotometry assay

Stocks

LB medium

TSS buffer

Chloramphenicol stock solution