Difference between revisions of "Team:Stony Brook/Protocols"

 
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<div class="column full_size">
 
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<div align="center">
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<h2> Lab Protocols</h2>
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<h1 style="font-family: Georgia;"> Protocols & Experiments </h1>
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</div>
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</div>
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<div align="center">
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<br>
 
<hr>
 
<hr>
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<br>
 
</div>
 
</div>
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<div class="column half_size">
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<div align="center">
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<h3> Agarose Gel Preparation </h3>
 +
</div>
 +
<ul>
 +
<li> Add 50mL of 1X TAE buffer to a 500mL erlenmeyer flask </li>
 +
<li> Add 0.5g of 1% agarose </li>
 +
<li> Microwave for three 20 second intervals and cool slightly </li>
 +
<li> Add 1ul Ethidium Bromide </li>
 +
<li> Mix and pour into gel box. Add Comb </li>
 +
<li> Cool for 30 minutes </li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
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<h3> Nanodrop </h3>  
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<div align="center">
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<ol>
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<h3> YPD Media </h3>
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<li> Open "Nucleic Acids" </li>
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</div>
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<li> Drop water on platform. Close it and blank. </li>
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<ul>
−
<li> Blank system with the solvent used for protocol </li>
+
<li> <h6>For liquid media </h6></li>
−
<li> Drop 1ul of sample on platform, close, click "measure"</li>
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<ul>
−
<li> Should have two humps </li>
+
<li> Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask </li>
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</ol>  
+
<li> Autoclave on the liquid setting </li>
 +
<li> Add 50mL of 40% glucose and mix </li>
 +
</ul>
 +
<h6> For solid media</h6>
 +
<ul>
 +
<li> Add same reagents as liquid, plus 24g of Bacto agar </li>
 +
<li> Autoclave </li>
 +
<li>Stir on a magnetic stir plate and add 50ml of 40% glucose</li>
 +
<li> Pour into petri dishes </li>
 +
</ul>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<br>
 
</div>
 
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
−
<h3> Ligation </h3>  
+
<div align="center">
−
<ol>
+
<h3> LB Broth <h3>
−
<li><pre>  
+
</div>
−
10X DNA Ligase Buffer → 2ul
+
<p> Add items to flask: </p>
−
Vector DNA (4kb) → 50ng
+
<ul>
−
Insert DNA (1kb) → 37.5ng
+
<li> 5g Tryptone </li>
−
Nuclease-Free Water → fill to 20ul
+
<li> 2.5g Yeast Extract </li>
−
T4 DNA ligase → 1ul
+
<li> 5g NaCl </li>
−
</pre></li>
+
<li> H<sub>2</sub>O filled to 500mL </li>
−
<li> Mix by pipetting up and down</li>
+
<li> Mix with magnetic stir bar on low heat, autoclave for liquids </li>
−
<li> Incubate at 16°C overnight or room temp for 10 minutes </li>
+
</ul>
−
<li> Heat inactive (heat block) at 65°C for 10 minutes </li>
+
<br>
−
<li> Chill on ice and transform 1-5ul of the reaction into 50ul competent cells</li>
+
<br>
−
<li> Insert in order of increasing expensiveness </li>
+
<br>
−
</ol>
+
<div align="center">
 +
<h3> Nanodrop </h3>
 +
</div>
 +
<ul>  
 +
<li> Select Nucleic Acid </li>
 +
<li> Clean, drop 1ul water, press okay </li>
 +
<li> Blank with 1ul </li>
 +
<li> Measure with 1ul of substance </li>
 +
<li> 260/280 ratio should be near 2 </li>
 +
</ul>
 
</div>
 
</div>
  
 +
<div class="column half_size">
 +
<div align="center">
 +
<h3> LB Agar </h3>
 +
</div>
 +
<p> Add items to flask: </p>
 +
<ul>
 +
<li> 5g Tryptone </li>
 +
<li> 2.5g Yeast Extract </li>
 +
<li> 5g NaCl </li>
 +
<li> 7.5g Agar </li>
 +
<li> H<sub>2</sub>O filled to 500mL </li>
 +
<li> Mix with magnetic stir bar on low heat, autoclave for liquids </li>
 +
<br>
 +
<br>
 +
<br>
 +
<div align="center">
 +
<h3> NEB Monarch Nucleic Acid Purification Protocols </h3>
 +
<p> The following NEB kits were used and protocols can be found on their <a id="stuff" style: color="red" href="https://www.neb.com/monarch/monarch-nucleic-acid-purification-kits"> website. </a></p>
 +
</div>
 +
<ul>
 +
<li> Monarch Plasmid Miniprep Kit </li>
 +
<li> Monarch DNA Gel Extraction Kit </li>
 +
<li> Monarch PCR & DNA Cleanup Kit </li>
 +
</ul>
 +
</div>
  
 +
<div class="column full_size">
 +
<br>
 +
</div>
  
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h3> Digest </h3>
 +
<ul>
 +
<li> Add Nuclease free water to a 2ml test tube</li>
 +
<li> Add 10X buffer for restriction enzymes </li>
 +
<li> Add restriction Enzymes </li>
 +
<li> Add DNA using (Need 500ng of DNA for 25ul) </li>
 +
<li> Make negative controls without the enzymes</li>
 +
<li>  Make up volume with nuclease free water</li>
 +
<li>  Amounts of reagents listed below </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> 50ul Reaction </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Nuclease-free water </td>
 +
<td> Bring to volume </td>
 +
</tr>
 +
<tr>
 +
<td> 10X Buffer </td>
 +
<td> 5</td>
 +
</tr>
 +
<tr>
 +
<td> Restriction Enzymes </td>
 +
<td> 1 each</td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> Calculate volume for DNA </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> 10ul Reaction </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Nuclease-free water </td>
 +
<td> Bring to volume </td>
 +
</tr>
 +
<tr>
 +
<td> 10X Buffer </td>
 +
<td> 5</td>
 +
</tr>
 +
<tr>
 +
<td> Restriction Enzymes </td>
 +
<td> .5 each</td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> Calculate volume for DNA </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<br>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h3> Transformation </h3>
 +
</div>
 +
<ul>
 +
<li> Thaw competent E. coli cells on ice and pipette 50ul cells into a transformation tube</li>
 +
<li>Add 1-5ul containing 1pg-100ng of plasmid DNA and flick tube 4-5 times</li>
 +
<li> Place tube on ice for 30 minutes </li>
 +
<li> Heat shock at 42°C for 30 seconds </li>
 +
<li> Place on ice for 5 minutes </li>
 +
<li> Add 950ul room temperature SOC to mixture </li>
 +
<li> Incubate at 37°C for 60 minutes at 250rpm </li>
 +
<li> Mix and perform several 10-fold serial dilutions in SOC </li>
 +
<li> Spread 50-100ul onto a plate and incubate overnight at 37°C</li>
 +
</ul>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h3> Ligation </h3>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Nuclease-free water</td>
 +
<td> Bring to volume </td>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Ligase Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> T4 Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
<tr>
 +
<td> Vector : Insert Ratio </td>
 +
<td> 1:3</td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
<ul>
 +
<li>There must be 3x as much insert as vector </li>
 +
<li> Total volume of solution must be  20ul </li>
 +
<li> Negative control contains no inserts</li>
 +
</ul>
 +
</div>
 +
 +
<div class="column full_size">
 +
<br>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Phosphatase </h4>
 +
</div>
 +
<ol>
 +
<li> Add 1pmol of DNA ends </li>
 +
<li> Add 2ul of AP Buffer </li>
 +
</ol>
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<td> Content </td>
 +
<td> ul </td>
 +
</tr>
 +
<tr>
 +
<td> AP Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Phosphatase </td>
 +
<td> 1 ul</td>
 +
</tr>
 +
<tr>
 +
<td> Plasmid </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 7 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
  
  

Latest revision as of 07:49, 19 October 2016

Protocols & Experiments



Agarose Gel Preparation

  • Add 50mL of 1X TAE buffer to a 500mL erlenmeyer flask
  • Add 0.5g of 1% agarose
  • Microwave for three 20 second intervals and cool slightly
  • Add 1ul Ethidium Bromide
  • Mix and pour into gel box. Add Comb
  • Cool for 30 minutes

YPD Media

  • For liquid media
    • Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask
    • Autoclave on the liquid setting
    • Add 50mL of 40% glucose and mix
    For solid media
    • Add same reagents as liquid, plus 24g of Bacto agar
    • Autoclave
    • Stir on a magnetic stir plate and add 50ml of 40% glucose
    • Pour into petri dishes

LB Broth

Add items to flask:

  • 5g Tryptone
  • 2.5g Yeast Extract
  • 5g NaCl
  • H2O filled to 500mL
  • Mix with magnetic stir bar on low heat, autoclave for liquids



Nanodrop

  • Select Nucleic Acid
  • Clean, drop 1ul water, press okay
  • Blank with 1ul
  • Measure with 1ul of substance
  • 260/280 ratio should be near 2

LB Agar

Add items to flask:

  • 5g Tryptone
  • 2.5g Yeast Extract
  • 5g NaCl
  • 7.5g Agar
  • H2O filled to 500mL
  • Mix with magnetic stir bar on low heat, autoclave for liquids



    • NEB Monarch Nucleic Acid Purification Protocols

    The following NEB kits were used and protocols can be found on their website.

    • Monarch Plasmid Miniprep Kit
    • Monarch DNA Gel Extraction Kit
    • Monarch PCR & DNA Cleanup Kit

Digest

  • Add Nuclease free water to a 2ml test tube
  • Add 10X buffer for restriction enzymes
  • Add restriction Enzymes
  • Add DNA using (Need 500ng of DNA for 25ul)
  • Make negative controls without the enzymes
  • Make up volume with nuclease free water
  • Amounts of reagents listed below

50ul Reaction

    Content ul
    Nuclease-free water Bring to volume
    10X Buffer 5
    Restriction Enzymes 1 each
    DNA Calculate volume for DNA

10ul Reaction

    Content ul
    Nuclease-free water Bring to volume
    10X Buffer 5
    Restriction Enzymes .5 each
    DNA Calculate volume for DNA

Transformation

  • Thaw competent E. coli cells on ice and pipette 50ul cells into a transformation tube
  • Add 1-5ul containing 1pg-100ng of plasmid DNA and flick tube 4-5 times
  • Place tube on ice for 30 minutes
  • Heat shock at 42°C for 30 seconds
  • Place on ice for 5 minutes
  • Add 950ul room temperature SOC to mixture
  • Incubate at 37°C for 60 minutes at 250rpm
  • Mix and perform several 10-fold serial dilutions in SOC
  • Spread 50-100ul onto a plate and incubate overnight at 37°C

Ligation

Content ul
Nuclease-free water Bring to volume
10X T4 Ligase Buffer 2
T4 Ligase 1
Vector : Insert Ratio 1:3
  • There must be 3x as much insert as vector
  • Total volume of solution must be 20ul
  • Negative control contains no inserts

Phosphatase

  1. Add 1pmol of DNA ends
  2. Add 2ul of AP Buffer
Content ul
AP Buffer 2
Phosphatase 1 ul
Plasmid 10
H2O 7