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− | {{Stony_Brook}} | + | {{Stony_Brook TryingFailing}} |
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+ | <head> | ||
+ | <style> | ||
+ | ul { list-style-position: inside;} | ||
+ | li { list-style-position: inside; } | ||
+ | #stuff {color:#B61C1D; } | ||
+ | h1, h2, h3, h4, h5, h6 { font-family: Georgia;} | ||
+ | h1 { font-family: Georgia;} | ||
+ | </style> | ||
+ | </head> | ||
+ | <body> | ||
<div class="column full_size"> | <div class="column full_size"> | ||
<div align="center"> | <div align="center"> | ||
− | <h1> Protocols & Experiments </h1> | + | <h1 style="font-family: Georgia;"> Protocols & Experiments </h1> |
</div> | </div> | ||
</div> | </div> | ||
− | <div | + | <div align="center"> |
+ | <br> | ||
<hr> | <hr> | ||
+ | <br> | ||
</div> | </div> | ||
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<div class="column half_size"> | <div class="column half_size"> | ||
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<li> Microwave for three 20 second intervals and cool slightly </li> | <li> Microwave for three 20 second intervals and cool slightly </li> | ||
<li> Add 1ul Ethidium Bromide </li> | <li> Add 1ul Ethidium Bromide </li> | ||
− | <li> Mix and pour into gel box. Add Comb <li> | + | <li> Mix and pour into gel box. Add Comb </li> |
<li> Cool for 30 minutes </li> | <li> Cool for 30 minutes </li> | ||
</ul> | </ul> | ||
Line 40: | Line 47: | ||
<ul> | <ul> | ||
<li> Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask </li> | <li> Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask </li> | ||
− | <li> Autoclave on the liquid setting <li> | + | <li> Autoclave on the liquid setting </li> |
<li> Add 50mL of 40% glucose and mix </li> | <li> Add 50mL of 40% glucose and mix </li> | ||
</ul> | </ul> | ||
− | + | <h6> For solid media</h6> | |
<ul> | <ul> | ||
<li> Add same reagents as liquid, plus 24g of Bacto agar </li> | <li> Add same reagents as liquid, plus 24g of Bacto agar </li> | ||
Line 76: | Line 83: | ||
<ul> | <ul> | ||
<li> Select Nucleic Acid </li> | <li> Select Nucleic Acid </li> | ||
− | <li> Clean, drop 1ul water, | + | <li> Clean, drop 1ul water, press okay </li> |
− | <li> Blank </li> | + | <li> Blank with 1ul </li> |
<li> Measure with 1ul of substance </li> | <li> Measure with 1ul of substance </li> | ||
− | <li> | + | <li> 260/280 ratio should be near 2 </li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 100: | Line 107: | ||
<div align="center"> | <div align="center"> | ||
<h3> NEB Monarch Nucleic Acid Purification Protocols </h3> | <h3> NEB Monarch Nucleic Acid Purification Protocols </h3> | ||
− | <p> The following NEB kits were used and protocols can be found on their <a href="https://www.neb.com/monarch/monarch-nucleic-acid-purification-kits"> website </a></p> | + | <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> | </div> | ||
<ul> | <ul> | ||
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</div> | </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"> | <div class="column full_size"> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
<div class="column half_size"> | <div class="column half_size"> | ||
− | < | + | <div align="center"> |
+ | <h3> Transformation </h3> | ||
+ | </div> | ||
<ul> | <ul> | ||
− | <li>< | + | <li> Thaw competent E. coli cells on ice and pipette 50ul cells into a transformation tube</li> |
− | <li>< | + | <li>Add 1-5ul containing 1pg-100ng of plasmid DNA and flick tube 4-5 times</li> |
− | <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> | </ul> | ||
</div> | </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> | </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> | ||
+ | |||
+ | |||
+ | |||
</html> | </html> |
Latest revision as of 03:06, 12 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
- 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
For solid media
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
- Monarch Plasmid Miniprep Kit
- Monarch DNA Gel Extraction Kit
- Monarch PCR & DNA Cleanup Kit
NEB Monarch Nucleic Acid Purification Protocols
The following NEB kits were used and protocols can be found on their website.
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
- Add 1pmol of DNA ends
- Add 2ul of AP Buffer
Content | ul |
AP Buffer | 2 |
Phosphatase | 1 ul |
Plasmid | 10 |
H2O | 7 |