Team:CIDEB-UANL Mexico/WetLab-Protocols

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<p> LAB PROTOCOL
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IGEM-CIDEB_UANL </p>
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<link href='http://fonts.googleapis.com/css?family=PT+Sans+Narrow' rel='stylesheet' type='text/css' />
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#title {height: 55px; padding-left: 10px; background-color: #886A08; font-size:50px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
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#subtitle {height: 35px; padding-left: 10px; background-color: #DBA901; font-size:30px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
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<body>
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<table id="title" width="100%">
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<tr>
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<td>
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<div class="Estilo6">
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Wet-Lab</div>
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</table>
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<table id="subtitle" width="100%">
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<div class="Estilo6">
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Protocols
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<td>
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<p> <b>General Rules:</b>
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<br>1. - Always use your lab coat
 +
<br>2. - Always pick up the used material
 +
<br>3. - Never take any material to your mouth while you’re working with reactants
 +
<br>4. - Keep your working area clean
 +
<br>5. - All non-reusable material must be placed in the correct trash can
 +
<br>6. - Check for oppen gas and water valves, depending on your work
 +
<br>7. - Wash your hands before and after every session
 +
<br>8. - Always wear gloves when working with Ethidium bromide
 +
<br>9. - Every question, accident, etc. tell the instructor</p>
 +
 
 +
 
 +
<p><b>Recommendations for Microbiology</b>
 +
 
 +
<br>10. - Sterilize with heat any crop material before and after using it.
 +
<br>11. - Don’t talk while you are working with sterilized material
 +
<br>12. - When you start and finish, sterilize the surface with 70% alcohol
 +
<br>13. - Al testing tubes, must be vertical 
 +
<br>14. - Never deposit a living cultivation in the sink
 +
<br>15. - Sterilize every used material</p></td>
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<td>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/d/da/Imagen2.jpg" width="202px" height="203px" /> </p>
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</td>
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</tr></table>
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<td>
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<p> <b>Laboratory Protocol </b></p>
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<p><b> Handling of lyophilized DNA’s plates.</b> </p>
 +
 
 +
<p> This step is for searching and obtaining the desired piece.
 +
<br>Procedure:
 +
<br>1.- Search for the BioBrick in the Parts Registry 
 +
<br>2.- Drill the plate, and add 10ul of mQ water
 +
<br>3.- Mix very well, and take the resusupended DNA to an Eppendorf tube </p>
 +
 
 +
 
 +
<p> <b>Preparation for competent cells and their transformation</b></p>
 +
 
 +
<p>This is to prepare the plasmid in the bacterium with the thermal shock
 +
<br>Transformation Procedure:
 +
 
 +
<br>1.- Take 100ul of the bacteria to an Eppendorf tube
 +
<br>2.- Add 2ul of DNA and mix them
 +
<br>3.- Let in ice from 20 to 30 minutes
 +
<br>4.- Dive the Eppendorfs with 42°C water for 1 minute
 +
<br>5.- Take it to ice for 2 minutes
 +
<br>6.- Add LB and incubate them for 20 min, then put them into petri dish at 37°C all the night. </p></td>
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<td style="padding:12px;">
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/2/2a/Imagen1.jpg" width="266px" height="265px" /> </p>
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</td></tr></table>
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<td>
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<td>
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<p><b> Inoculation in Petri Dish and Test Tube </b></p>
 +
 
 +
<p>Inoculate bacteria in dishes with agar and medium tubes with their antibiotics
 +
<br>Procedure:
 +
 
 +
<br><b>For planting</b>
 +
<br>1.- Take the colonies in a test tube with LB and the antibiotic
 +
<br>2.- Take the handle sterilized and spread the colonies in a Petri dish.
 +
<br>3.- Incubate for 37°C FOR 16 hours
 +
<br>Reseeding
 +
<br>1.- Add the antibiotic to a test tube previously half filled with Lb medium
 +
<br>2.- With a handle take a colony
 +
<br>3.- Then agitate the handle in the Tube, finally incubate at 37°C </p>
 +
</td>
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<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/8/85/Imagen4.jpg" width="224px" height="227px" /> </p></td></tr></table>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/b/b4/Imagen3.jpg" width="500px" height="200px" /> </p>
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<tr>
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<td>
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<p><b>Plasmid DNA miniprep</p></b>
 +
 
 +
<p>This is to obtain the DNA plasmid of a colony
 +
<br>Procedure:
 +
 
 +
<br>1.- Add 1.5ml of colonies to an Eppendorf, centrifuge 30 seconds and throw the liquid  in the trash with soap.
 +
<br>2.- Add  200ul of solution I, II and III in times of 5 minutes respectively and then centrifuge the new solution.
 +
<br>3.- Pass the liquid to and Eppendorf containing 1ml of alcohol at 100%
 +
<br>4.- Incubate at -20°C for 10 minutes
 +
<br>5.- Centrifuge and throw the liquid.
 +
<br>6.- Add 200ul of Ethanol 70% and mix very well
 +
<br>7.- Centrifuge again and throw the liquid
 +
<br>8.- Let the precipitate incubate at 37°C, then add 200ul of mQ water with RNase and mix it
 +
<br>9.- Check in the gel or save it at 4°C </p></td>
 +
<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/e/e6/Imagen_1.jpg" width="132px" height="285px" /> </p></td></tr></table>
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<table style="background-color: #FFFFFF;" width="100%" id="texto">
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<tr>
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<td>
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<p><b> DNA quantification by UV spectrophotometer</b> </p>
 +
 
 +
<p>This is for quantify the plasmid DNA extracted
 +
<br> Procedure:
 +
 
 +
<br>1.- Take 1ml of mQ water in an Eppendorf
 +
<br>2.- Add 1ul of plasmid DNA
 +
<br>3.- Prepare the spectrophotometer
 +
<br>4.- Pass the DNA to the spectrophotometer
 +
<br>5.- Select the option (DNA or RNA)
 +
<br>6.- Write the lecture of the spectro at 260, 280 and 320, relation 260/280 and the concentration.</p></td></tr></table>
 +
 
 +
<table style="background-color: #FFFFFF;" width="100%" id="texto">
 +
<tr>
 +
<td>
 +
<p> <b>Plasmid DNA characterization </b></p>
 +
 
 +
<p> This is for identify the piece we have
 +
<br>Procedure:
 +
 
 +
<br>1.- Prepare the mix
 +
<br>2.- Deal the mix in equal parts
 +
<br>3.- Add the DNA and mix
 +
<br>4.- Incubate the reactions at 37°C for 1 hour
 +
<br>5.- Use the gel for check the result </p>
 +
</td>
 +
<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/a/af/Imagen6.jpg" width="282px" height="232px" /> </p></td></tr></table>
 +
<p><b>BioBrick parts assembly</b></p>
 +
 
 +
<p> Procedure:
 +
 +
<br>In this step we are going to cut the plasmid with the appropriate restriction enzymes for deliver the desired fragment and to paste into another bacterium.
 +
<br>1.- Prepare the mix
 +
<br>2.- Lead the mix in equal parts and the add DNA, mix it.
 +
<br>3.- Incubate the reactions for 1 hour
 +
<br>4.- Check in the gel
 +
<br>5.- Save it for its ligation</p>
 +
 
 +
 
 +
<p><b>Ligation of genetic parts</b></p>
 +
 
 +
<p>This step is for paste the desired piece in the bacterium we are going to use.
 +
<br>Procedure:
 +
 
 +
<br>1.- Take in count the concentration of each one of the DNA’s.
 +
<br>2.- Use the calculator for obtain the amounts for preparing the ligation mix at 20ul
 +
<br>3.- Prepare the mix in the next order: mQ water, Buffer, and the plasmid
 +
<br>4.- Lead the mix and add the appropriate ligase
 +
<br>5.- Incubate the reactions at 25°C</p>
 +
 
 +
</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 
 +
</body>
 +
 
 +
</html>
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{{:Team:CIDEB-UANL_Mexico/footer}}

Latest revision as of 01:14, 22 June 2013

Wet-Lab
Protocols

General Rules:
1. - Always use your lab coat
2. - Always pick up the used material
3. - Never take any material to your mouth while you’re working with reactants
4. - Keep your working area clean
5. - All non-reusable material must be placed in the correct trash can
6. - Check for oppen gas and water valves, depending on your work
7. - Wash your hands before and after every session
8. - Always wear gloves when working with Ethidium bromide
9. - Every question, accident, etc. tell the instructor

Recommendations for Microbiology
10. - Sterilize with heat any crop material before and after using it.
11. - Don’t talk while you are working with sterilized material
12. - When you start and finish, sterilize the surface with 70% alcohol
13. - Al testing tubes, must be vertical
14. - Never deposit a living cultivation in the sink
15. - Sterilize every used material

Laboratory Protocol

Handling of lyophilized DNA’s plates.

This step is for searching and obtaining the desired piece.
Procedure:
1.- Search for the BioBrick in the Parts Registry
2.- Drill the plate, and add 10ul of mQ water
3.- Mix very well, and take the resusupended DNA to an Eppendorf tube

Preparation for competent cells and their transformation

This is to prepare the plasmid in the bacterium with the thermal shock
Transformation Procedure:
1.- Take 100ul of the bacteria to an Eppendorf tube
2.- Add 2ul of DNA and mix them
3.- Let in ice from 20 to 30 minutes
4.- Dive the Eppendorfs with 42°C water for 1 minute
5.- Take it to ice for 2 minutes
6.- Add LB and incubate them for 20 min, then put them into petri dish at 37°C all the night.

Inoculation in Petri Dish and Test Tube

Inoculate bacteria in dishes with agar and medium tubes with their antibiotics
Procedure:
For planting
1.- Take the colonies in a test tube with LB and the antibiotic
2.- Take the handle sterilized and spread the colonies in a Petri dish.
3.- Incubate for 37°C FOR 16 hours
Reseeding
1.- Add the antibiotic to a test tube previously half filled with Lb medium
2.- With a handle take a colony
3.- Then agitate the handle in the Tube, finally incubate at 37°C

Plasmid DNA miniprep

This is to obtain the DNA plasmid of a colony
Procedure:
1.- Add 1.5ml of colonies to an Eppendorf, centrifuge 30 seconds and throw the liquid in the trash with soap.
2.- Add 200ul of solution I, II and III in times of 5 minutes respectively and then centrifuge the new solution.
3.- Pass the liquid to and Eppendorf containing 1ml of alcohol at 100%
4.- Incubate at -20°C for 10 minutes
5.- Centrifuge and throw the liquid.
6.- Add 200ul of Ethanol 70% and mix very well
7.- Centrifuge again and throw the liquid
8.- Let the precipitate incubate at 37°C, then add 200ul of mQ water with RNase and mix it
9.- Check in the gel or save it at 4°C

DNA quantification by UV spectrophotometer

This is for quantify the plasmid DNA extracted
Procedure:
1.- Take 1ml of mQ water in an Eppendorf
2.- Add 1ul of plasmid DNA
3.- Prepare the spectrophotometer
4.- Pass the DNA to the spectrophotometer
5.- Select the option (DNA or RNA)
6.- Write the lecture of the spectro at 260, 280 and 320, relation 260/280 and the concentration.

Plasmid DNA characterization

This is for identify the piece we have
Procedure:
1.- Prepare the mix
2.- Deal the mix in equal parts
3.- Add the DNA and mix
4.- Incubate the reactions at 37°C for 1 hour
5.- Use the gel for check the result

BioBrick parts assembly

Procedure:
In this step we are going to cut the plasmid with the appropriate restriction enzymes for deliver the desired fragment and to paste into another bacterium.
1.- Prepare the mix
2.- Lead the mix in equal parts and the add DNA, mix it.
3.- Incubate the reactions for 1 hour
4.- Check in the gel
5.- Save it for its ligation

Ligation of genetic parts

This step is for paste the desired piece in the bacterium we are going to use.
Procedure:
1.- Take in count the concentration of each one of the DNA’s.
2.- Use the calculator for obtain the amounts for preparing the ligation mix at 20ul
3.- Prepare the mix in the next order: mQ water, Buffer, and the plasmid
4.- Lead the mix and add the appropriate ligase
5.- Incubate the reactions at 25°C
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