Team:Sevilla/Week4

From 2011.igem.org

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4. 72ºC for 3 minutes.</p>
4. 72ºC for 3 minutes.</p>
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<p>Steps 2 to 4 must be repeated for 30 times</p>
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Latest revision as of 22:28, 21 September 2011


Monday 1 August


The primers we ordered last week should have come today, but as usual, they haven't (bureaucracy, lazy postmen, maybe a natural catastrophe...). This means we can't do much today, except plan our next steps and search for more information about how to build logic gates, or decipher the intrincate registry of parts. We've realised we have some useful biobricks in our distribution kit which we didn't even know about.

Another useful way of spending time: preparing the videos for the people who gave us money through the crowdfunding platform, in appreciation for their donations.

Even more useful: joining together pipette tips to build a giant ring!


Monday Protocols



Tuesday 2 August


Our primers still haven't arrived, but happily, to ease our boredom the university's cabling decides to short-circuit! Our incubator starts steaming, the fire alarm starts ringing, the fridges don't work, our frozen cultures start to melt, but we don't panic...until we find out the Internet doesn't work either!!!


Tuesday Protocols



Wednesday 3 August


More mini-preps, more transformations, and more competent cells to kill the time while the primers arrive, but at least we're improving and the results are pretty good! Besides, we're trying out a new protocol to purify DNA after digestions. Tomorrow we'll see how it goes.


Wednesday Protocols


Ethanol Purification

-MATERIALS

1. Absolute Ethanol (100%) at -20ºC.

2. Ethanol 95% at -20ºC.

3. Temperature regulated centrifuge.

4. Feezer at -80ºC.

5. Milli-Q water

6. Sodium Acetate 3M.

7. Ice.

PROTOCOL

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.

2. Add 10μL of Sodium Acetate (only if the sample´s colume is 90μL,as it must be).

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).

4. Centrifugate for 30 minutes at 0ºC at máximum speed (>10000g).

5. Discart 70μL of supernatant. You may use a pipette without touching the eppendorf bottom.

6. Add 800μL of cold etanol 95% and invert the tubes once or twice inmediately. After that, incubate for 1 h at -80ºC.

7. Centrifugate at 4ºC for 10 minutes at máximum speed(>10000g).

8. Discart 770μL of supernatant. You may use a pipette without touching the eppendorf bottom.

9. Let the pellet dry. (It´s very important that the pellet is completely dry)You can use the stove at 37ºC.

10. Add 10μL of Milli-Q wáter to the sample.


Thursday 4 August


After purifications we run an electrophoresis and it turns out so beautifully we almost cry! Everything has gone as expected, the digestions are ok, but the NanoDrop results say we've lost too much DNA in the process.

The primers are here at last! We can finally begin the PCR reactions to regenerate plasmid backbones and obtain some genes from our donated strains. Let's see how we go...


Thursday Protocols


PCR PROTOCOL

We use the Maxime Premix kit to do our PCR. Maxime PCR PreMix contains:

- i-MAX (II)DNA Polymerase.

- dNTPs.

- Reaction Buffer

- Gel Loading buffer.

We only have to add 17 μL, 1 μL of DNA sample and 1 μL of each primers (Foward and Reverse)to the premix.

PCR cicle

1. 94ºC for 3 minutes.

2. 94ºC for 30 seconds.

3. 55ºC for 30 seconds.

4. 72ºC for 3 minutes.

.

Steps 2 to 4 must be repeated for 30 times


Friday 5 August


Roberto has brought M&M's: lots of them!!!This is the best day of our lives. Besides, the PCR results are really quite good, except for the fact that we've amplified the wrong BioBrick - it seems working here for so many hours has overheated our brains. We'll have to try again next week, and make sure we pay more attention.


Weekend


We have a bit of a do to celebrate Roberto's birthday. Look how elegant we can be on special occasions!

We also spend the whole Sunday watching a "Game of Thrones" marathon at Marta's while eating pizza. What an awsome series! Highly recommended by Arcanum - you can't miss it!

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