Team:Sevilla/Week5

From 2011.igem.org

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<p>Purification of the digestions with ethanol before ligations has proven to be important, but we still don't get the protocol right. Give us some time to optimize it!</p>
<p>Purification of the digestions with ethanol before ligations has proven to be important, but we still don't get the protocol right. Give us some time to optimize it!</p>
<p>However some good news, the second PCR has been a complete success. We have to purify the DNA from the gel - a new protocol should be exciting, but it's becoming more like a nightmare!</p>
<p>However some good news, the second PCR has been a complete success. We have to purify the DNA from the gel - a new protocol should be exciting, but it's becoming more like a nightmare!</p>
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<img src="https://lh5.googleusercontent.com/-Yn6Lnl3nW8Y/Tnornwj7ZdI/AAAAAAAAA6Y/86wd3kX5I-Y/s288/la%252520foto%2525206.JPG" style=padding-left:200px;" height="215" width="288" /></a>
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<input type="button" value="Show Monday protocols" id="mondayBP">
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<strong>GenElute Gel Extraction Kit from SIGMA</strong>
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<strong>Purification of DNA from agarose gel</strong>
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<p>Purification of DNA from agarose gel.</p>
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<p>GenElute Gel Extraction Kit from SIGMA</p>
<p>1.Excise band fron the agarose gel and weigh the gel</p>
<p>1.Excise band fron the agarose gel and weigh the gel</p>
<p>2.For every 100 mg of agarose gel, add 300 μl of Gel Solubilization Solution. Incubate the gel mixture at 55ºC for 10 minutes, or until the gel slice is completely dissolved. Vortex every 2-3 minutes.</p>
<p>2.For every 100 mg of agarose gel, add 300 μl of Gel Solubilization Solution. Incubate the gel mixture at 55ºC for 10 minutes, or until the gel slice is completely dissolved. Vortex every 2-3 minutes.</p>
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<p>6.Add 700 μl of Wash Solution to the binding column. Centrifuge for 1 minute. Discard the flow-through and repeat this step to remove excess ethanol.</p>
<p>6.Add 700 μl of Wash Solution to the binding column. Centrifuge for 1 minute. Discard the flow-through and repeat this step to remove excess ethanol.</p>
<p>7.Transfer the binding column to a fresh collection tube. Add 30 μl of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 min.</p>
<p>7.Transfer the binding column to a fresh collection tube. Add 30 μl of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 min.</p>
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<strong>Ethanol Purification</strong>
 
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<p>-MATERIALS</p>
 
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1. Absolute Ethanol  (100%) at -20ºC.
 
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2. Ethanol  95%  at  -20ºC.
 
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3. Temperature regulated centrifuge.
 
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4. Feezer at -80ºC.
 
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5. Milli-Q water
 
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6. Sodium Acetate 3M.
 
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7. Ice.
 
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PROTOCOL
 
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1. Add 2 volumes of very cold absolute etanol (preserved at -20ºC) to the sample (the sample must have 30μL, so you must add  60μL of ethanol). Hacer este paso y el siguiente en hielo.
 
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2. Add  10μL of Sodium Acetate (only if the sample´s colume is  90μL,as it must be).
 
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3. Incubate for 1h a -80ºC (it can be more time if the DNA fragments are very little). It can also be an overnight incubation).
 
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4. Centrifugate for 30 minutes at 0ºC at  máximum speed (>10000g).
 
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5. Discart  70μL of supernatant. You may use a pipette without touching the eppendorf bottom.
 
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6. Add  800μL of cold etanol 95% and invert the tubes once or twice inmediately. After that, incubate for  1 h at -80ºC.
 
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7. Centrifugate at 4ºC for 10 minutes at máximum speed(>10000g).
 
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8. Discart  770μL of supernatant. You may use a pipette without touching the eppendorf bottom.
 
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9. Let the pellet dry. (It´s very important that the pellet is completely dry)You can use the stove at 37ºC.
 
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10. Add  10μL  of Milli-Q wáter to the sample.
 
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
 
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
 
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<input type="button" value="Show Wednesday protocols" id="wednesdayBP">
 
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
 
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed p>pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
 
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The department of Genetics is hardly accessible at all today, but things aren't any better here in the lab: our fridge doesn't work properly and now we have all our chemicals and DNA samples surrounded by blocks of ice.  
The department of Genetics is hardly accessible at all today, but things aren't any better here in the lab: our fridge doesn't work properly and now we have all our chemicals and DNA samples surrounded by blocks of ice.  
</p>
</p>
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<img src="https://lh5.googleusercontent.com/-bdb-c3WMdlM/Tno7MKj6XaI/AAAAAAAAA6k/K2O84jQMmCs/s288/DSCF6315.jpg" style=padding-left:350px;" height="216" width="288" /></a>
<p> How to fix a freezer, lesson one: gather some mops, ice trays to keep things cool and something to crush the blocks until they come unstuck. The rest is easy: open the freezer door, put your chemicals in the trays, and then charge, charge, charge at the blocks and let the ice melt. Then clean up all the mess and voila, you'll have plenty of space for all your digestions, biobricks, etc.
<p> How to fix a freezer, lesson one: gather some mops, ice trays to keep things cool and something to crush the blocks until they come unstuck. The rest is easy: open the freezer door, put your chemicals in the trays, and then charge, charge, charge at the blocks and let the ice melt. Then clean up all the mess and voila, you'll have plenty of space for all your digestions, biobricks, etc.
</p>
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<input type="button" value="Show Thursday protocols" id="thursdayBP">
 
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<div id="thursdayProtocols">
 
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<p>Thursday Protocols</p>
 
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
 
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
 
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We finally got some ligations right, but they're too few and we're not sure whether they're OK. We'll have to study them carefully...
We finally got some ligations right, but they're too few and we're not sure whether they're OK. We'll have to study them carefully...
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</p>
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<input type="button" value="Show Friday protocols" id="fridayBP">
 
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<p>Friday Protocols</p>
 
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
 
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
 
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<div id="weekend">
 
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<p>Weekend</p>
 
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</html>{{:Team:Sevilla/Templates/hr|colour=#9B162B|class=weekend1}}<html>
 
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<div class="summary">
 
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
 
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
 
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<input type="button" value="Show Week" class="SW" style="margin-bottom: 10px;z-index:4;">
 
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Latest revision as of 01:42, 22 September 2011


Monday 8 August


Purification of the digestions with ethanol before ligations has proven to be important, but we still don't get the protocol right. Give us some time to optimize it!

However some good news, the second PCR has been a complete success. We have to purify the DNA from the gel - a new protocol should be exciting, but it's becoming more like a nightmare!


Monday Protocols


Purification of DNA from agarose gel

GenElute Gel Extraction Kit from SIGMA

1.Excise band fron the agarose gel and weigh the gel

2.For every 100 mg of agarose gel, add 300 μl of Gel Solubilization Solution. Incubate the gel mixture at 55ºC for 10 minutes, or until the gel slice is completely dissolved. Vortex every 2-3 minutes.

3.Prepare binding column. Place the GenElute Binding Column G into one of 2 ml collection tubes. Add 500 μl of the Column Preparation Solution to each binding column. Centrifuge for 1 minute.

4.Add 1 gel volume of 100% isopropanol and mix until homogenous.

5.Load the solubilized gel solution mixture into the binding column. Centrifuge 1 min. Discard the flow-through

6.Add 700 μl of Wash Solution to the binding column. Centrifuge for 1 minute. Discard the flow-through and repeat this step to remove excess ethanol.

7.Transfer the binding column to a fresh collection tube. Add 30 μl of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 min.



Tuesday 9 August


The purifications from gel didn't go as expected, and to make matters worse the whole department of Genetics is in a complete chaos; there's no floor, all the important machines are hidden, and we don't have access to most of the rooms. It seems it won't get any better during the week...


Wednesday 10 August


A freshly cemented corridor separates us from the door behind which our PCR reactions are waiting for us to rescue them. As well as this, getting to the NanoDrop or the Transiluminator rooms has become an adventure due to the piles of rubble and the hanging wires we have to dodge. This department has become a modern jungle, so we try not to come over here, and stay in our downstairs lab working on more digestiones and electrophoresis.


Thursday 11 August


The department of Genetics is hardly accessible at all today, but things aren't any better here in the lab: our fridge doesn't work properly and now we have all our chemicals and DNA samples surrounded by blocks of ice.

How to fix a freezer, lesson one: gather some mops, ice trays to keep things cool and something to crush the blocks until they come unstuck. The rest is easy: open the freezer door, put your chemicals in the trays, and then charge, charge, charge at the blocks and let the ice melt. Then clean up all the mess and voila, you'll have plenty of space for all your digestions, biobricks, etc.


Friday 12 August


We finally got some ligations right, but they're too few and we're not sure whether they're OK. We'll have to study them carefully...

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