Team:Sevilla/Week10

From 2011.igem.org

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<p>Keep at room temperature overnight.</p>
<p>Keep at room temperature overnight.</p>
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<p style="text-decoration:underline;">Double Digestion</p>
<p style="text-decoration:underline;">Double Digestion</p>
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<p>- 1 μL of DNA sample</p>
<p>- 1 μL of DNA sample</p>
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<p>It´s important that you shake the mixture using the centriguge. 1 minute at medium speed must works.</p>
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<p>It´s important that you shake the mixture using the centrifuge. 1 minute at medium speed must works.</p>
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</br>
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<p style="text-decoration:underline;">Transformtion</p>
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<p>1. In the same tube, mix:</p>
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<p>- 25 μL of competent cells. </p>
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<p>- 1 μL of DNA sample (the amount of DNA depends on the concentration of your sample)if yor DNA is pure. If not, use aproximately 3 μL. </p>
 +
<p>*Use very cold pipets.</p>
 +
<p>2. Incubate the tube on ice for 30 minutes.</p>
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<p>3. Incubate at 42ºC for 30 seconds.</p>
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<p>4. Incubete 2 minutes on ice for 2 minutes.p>
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<p>5. Add 225 μL of NZY, LB, or SOB (preheated in the 42 degrees bath). You must add 225 μL of medium culture per each microlitre of DNA sample you use.</p>
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<p>6. Keep the tube for 45 minutes or 1h in agitacion at 37ºC
 +
 
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<p>**If you are using complez DNA ligations as for the transformation, you must add beta-mercaptoethanol before the half an hour incubation on ice</p>
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Ligation 1 has grown!!!! Isn't it incredible? It's a fusion of F2620 and I732019, it should be able to sense C6-3-oxo-HSL in the media and express lacZ as a reporter. We'll have to make a liquid culture to get the plasmid tomorrow, digest it to check if the insert has the appropiate length and see if it works. For the last part, we seed the bacteria in a plate with the signal and XGal that should turn blue when lacZ is expressed.  
Ligation 1 has grown!!!! Isn't it incredible? It's a fusion of F2620 and I732019, it should be able to sense C6-3-oxo-HSL in the media and express lacZ as a reporter. We'll have to make a liquid culture to get the plasmid tomorrow, digest it to check if the insert has the appropiate length and see if it works. For the last part, we seed the bacteria in a plate with the signal and XGal that should turn blue when lacZ is expressed.  
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<img src="https://lh3.googleusercontent.com/--RkNqT7OJ98/Tno8OfwDBuI/AAAAAAAAA64/208ixYpxK68/s640/DSCF6321.jpg" style=padding-left:250px;"height="288" width="192" />
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<p>On the other hand, our constructions haven't arrived on time so we'll have to give up that option and stick to what we have now. We're having a bussy weekend: the parts must be sent next week and there's still a lot to do.LAST PUSH GUYS!
<p>On the other hand, our constructions haven't arrived on time so we'll have to give up that option and stick to what we have now. We're having a bussy weekend: the parts must be sent next week and there's still a lot to do.LAST PUSH GUYS!
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<img src="https://lh3.googleusercontent.com/-baIqMTNu82A/Tnpmd1PVHpI/AAAAAAAAA8Y/MiRlr9JEItI/s640/DSCF6325.jpg" style=padding-left:150px;"height="188" width="192" /> <img src="https://lh4.googleusercontent.com/-lFxGFCxmJaE/TnpmelpHmDI/AAAAAAAAA8g/2QrdJFJBGrA/s640/DSCF6326.jpg" height="170" width="192" />
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<p> We're taking turns to get some sleep, but the floor is not exactly comfortable, and the cleaners come at 6 a.m... At least we have enough food to survive, a coffee machine and 4 litres of Coca-Cola. What a long weekend...</p>
<p> We're taking turns to get some sleep, but the floor is not exactly comfortable, and the cleaners come at 6 a.m... At least we have enough food to survive, a coffee machine and 4 litres of Coca-Cola. What a long weekend...</p>
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<img src="https://lh6.googleusercontent.com/-XqqyHKLwghM/TnpmkDaHaxI/AAAAAAAAA8w/E7YfaM3IyTo/s640/DSCF6330.jpg" style=padding-left:50px;"height="188" width="190" />
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Latest revision as of 01:06, 22 September 2011


Monday 12 September


One more week. We get to the lab in the morning. We open the incubator...

What the...FUCK? Honestly,is somebody kidding us? Where's the hidden camera? What the hells' wrong? Not a single colony in our transformation plates. AGAIN. Will it EVER work, for goodness sake? We've tried almost everything! Should we pray to God, to Superman, to Mr. Gibss?? Let's start again... :(


Monday Protocols


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. 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.


Tuesday 13 September


At least the boys are coming back little by little and we have more hands in the lab to work, and this is a bit more crowded and cheerful. We're starting to think more seriously about the Jamboree, there's still so much to do about the wiki, the parts, the team t-shirts, the flights, etc.


Tuesday Protocols


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. 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.


Wednesday 14 September


While we keep fighting against our naughty bacteria and the "standard" parts, we're also testing the biobrick T9002, developed by the Cambridge Lab in 2005. Ana is helping us to plan the experiments.


Wednesday Protocols


IMPROVED PROTOCOLS

Ligation

We are going to assemble small DNA fragments (inserts) with plasmids, so we will use bigger amount of inserts than os plasmid. Our ligation mix contains:

- 1 μL of 10X ligase buffer. It´s important to keep it on ice and also to resuspend it very well before using it. (Ligase buffer must contains ATP).

- 1 μL of ligase(It must be the last thing you add to the mix). You must not sink the tip too deep in order not to carry much glycerol. KEEP IT IN THE FREEZER, even when you use it.

- 1 or 2 μL of plasmid.

- 7 or 6 μL of insert.

Keep at room temperature overnight.


Double Digestion

20 μL must be the final volumen of our digestion mix. We must add:

- 2 μL of buffer

- 0,5 μL of each enzyme (We use EcorI, SpeI, XbaI and PstI). This amount of enzyme must be individually determine depending on the amunt of DNA you are going to digest

- 16 μL of Milli-Q water

- 1 μL of DNA sample


It´s important that you shake the mixture using the centrifuge. 1 minute at medium speed must works.


Transformtion

1. In the same tube, mix:

- 25 μL of competent cells.

- 1 μL of DNA sample (the amount of DNA depends on the concentration of your sample)if yor DNA is pure. If not, use aproximately 3 μL.

*Use very cold pipets.

2. Incubate the tube on ice for 30 minutes.

3. Incubate at 42ºC for 30 seconds.

4. Incubete 2 minutes on ice for 2 minutes.p>

5. Add 225 μL of NZY, LB, or SOB (preheated in the 42 degrees bath). You must add 225 μL of medium culture per each microlitre of DNA sample you use.

6. Keep the tube for 45 minutes or 1h in agitacion at 37ºC

**If you are using complez DNA ligations as for the transformation, you must add beta-mercaptoethanol before the half an hour incubation on ice


Thursday 15 September


Ligation 1 has grown!!!! Isn't it incredible? It's a fusion of F2620 and I732019, it should be able to sense C6-3-oxo-HSL in the media and express lacZ as a reporter. We'll have to make a liquid culture to get the plasmid tomorrow, digest it to check if the insert has the appropiate length and see if it works. For the last part, we seed the bacteria in a plate with the signal and XGal that should turn blue when lacZ is expressed.



Thursday Protocols


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. 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.


Friday 16 September


YEAAAAAAAAAH! IT WORKS!!! Look at this plate and the little blue spots! IT WORKS! Sadly,we have to digest the plasmid, extract the insert and place it in pSB1C3, because now it's in pSB1A2. Let's hope this time the cycle works as it should.

On the other hand, our constructions haven't arrived on time so we'll have to give up that option and stick to what we have now. We're having a bussy weekend: the parts must be sent next week and there's still a lot to do.LAST PUSH GUYS!


Friday Protocols


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. 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.


Weekend


Marathon weekend doing mini-preps of every single colony we have, with their restrictions, electrophoresis, purification, etc. This means more than 30 digestions ONLY for one construction. Besides, we're also working with some of the other planned constructions, and we're doing everything in duplicate to try different protocols in line. We're running out of time,and the electrophoresis machinery is smoking. We're using two different competent cells, kipping the ligationsat different temperatures at different times...We have to try everything!

We're taking turns to get some sleep, but the floor is not exactly comfortable, and the cleaners come at 6 a.m... At least we have enough food to survive, a coffee machine and 4 litres of Coca-Cola. What a long weekend...



Weekend Protocols


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. 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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