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Team:Sevilla/Week2
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Monday 18 July
Our inoculums look pleasantly cloudy, as expected. All except two. We freeze samples of the good ones in glycerol at -80 ºC in a wonderful fridge that could also easily house a corpse (who knows?).
We start studying the protocols of our mini-prep kit to purify the BioBrick plasmids from our transformed cultures. They don't look that complicated but we have to do about 50; shoulders to the wheel!
We receive several BioBricks and the strains we had ordered: vainillin and rhlR BioBricks, two strains of B.subtilis, another strain containing VanM/N genes... people are being quite kind to us! We seed the bacteria and transform them with the BioBricks, depending on the format they come in.
Our mini-preps are ready at last after a hard day’s work, and we are able to leave some inoculums of DH5-a to prepare competent cells tomorrow.
21.30: Lab OFF.
Monday Protocols
Miniprep Protocol:
We use the Miniprep Protocol giving in the kit of Favorgen biotech corp.:
1. Transfer 2 ml of well-grown bacteria culture to a microcentrifuge tube.
2. Descend the bacteria by centrifuging at 10.000 rpm for 1 minute and discard the supernatant completely.
3. Add 250 μl of FAPD1 Buffer to the pellet and resuspend the cells completely by pipetting.
4. Add 250 μl of FAPD2 Buffer and gently invert the tube 5 times to lyse the cells and incubate at room temperature for 2 min.
5. Add 350 μl of FAPD3 Buffer and invert the tube 5 times
6. Centrifuge for 10 min at 14.000 rpm
7. Transfer the suspernatant carefully to FAPD Column. Centrifuge for 1 min and then discard the flow-through.
8. Add 400 μl of W1 Buffer to FAPD Column. Centrifuge for 1 min then discard the flow-through.
9. Add 750 μl of Wash Buffer to FAPD Column. Centrifuge for 1 min then discard the flow-through.
10. Centrifuge for an additional 3 min to dry the column.
11. Place FAPD Column to a new 1,5 ml microcentrifuge tube.
12. Add 30 μl of ddH2O to the membrane center of FAPD Column. Stand the column for 2 min.
13. Centrifuge for 2 min at 14.000 rpm to elute plasmid DNA.
Tuesday 19 July
Materials and chemicals keep appearing in big cardboard boxes full of styro-foam pieces (we're storing it all in the hopes of filling a bath next month and having some fun).
More good news: our transformed cultures have grown as expected, as have the B. subtilis strains. At the end of the day we make inoculums of them all so that they grow o/n and tomorrow we can freeze samples and do yet more mini-preps (we have only just started and we're already sick and tired of that kit...)
We need to discuss which assembling method we'll use with the BioBricks. It takes us some hours, and in the meantime, Andrés faces the personal challenge of making competent cells from the DH5-a inoculum with the new protocol. Tomorrow we'll see who has won the battle...
And Pedro isn't wasting any time at all, he's just found out these racks are stackable, like Lego. This discovery has changed his life, look how happy he is!
After deciding how we'll build each BioBrick, and counting all the ligations we need to do, we finally tackle the digestions with the different restriction enzymes. Mastermix kits this time. More protocols. GREAT.
Tuesday Protocols
Células competentes II
1)Incubate a bacterial colony in 3 mL LB, 37ºC with agitation all night.
2)Take 300 μL from this LB and put it into another 30 mL LB medium at 37ºC until reaching an optical density of 0’35 – 0’6 at 600 wavelength
3)Take away 1 mL and cool on ice for 5 minutes.
4)Centrifuge for 30 seconds at 15000g, discard the supernatant and resuspend the pellet in 75 μL of cold LB and keep it in ice for 5 min.
5)Add 75 μL Solution TSS 2x, mix everything and keep it in ice for 5 min. Cells now can be used inmediately o freeze them at -80ºC.
Solution TSS contains: Triptone 8g/l, yeast extract 5 g/L, NaCl 5g/L, Polietilenglicol (PEG) 8000 200 g/L, DMSO 10% (v/v), 100ml/L MgSO4 1M. Add 700 mL of water and calibrate the pH to 6,5. Finally, fill it until reach 1L.
Wednesday 20 July
AT LAST our official mascot is here! Say hello to Mr. Gibbs! Rocío had him held captive in her house, but to make up for that, she's brought a WONDERFUL Disney princess clock to cheer our lab up! It gives the lab that homely touch. We have also brought a coffee machine - we need caffeine in industrial amounts to work here for so many hours a day.
More mini-preps and frozen samples. But we're also trying something new! Our first electrophoresis to see how the digestions and the mini-preps go. We're examining the results. It's ok for the first time, but there's a long way to go yet...
Marta is in charge of our first ligations, tomorrow we'll see how they went.
...and how did the great battle end? Andrés 1 - E.coli 0. We have competent cells!
Out of the lab before 19.00. What a record!
Wednesday Protocols
Digestion
1.Make the following master mix:
-1 μl enzyme 1
-1 μl enzyme 2
-3 μl enzyme buffer
-15 μl ddH2O
2.Add 20 μl of master mix to 10 μl of DNA
3.Incubate this mix 3 hours in a bath at 37ºC
4.Make a heat shock at 80 ºC for 20 min
5.Store the eppendorfs at 4ºC
Thursday 21 July
We run an electrophoresis of the digestions we made yesterday, but it hasn't worked properly, it seems the process still needs some fine-tuning.
We spend some time preparing the rewards for those who gave us money for the project through the crowd-funding platform. Letters, funny badges, videos...
Coffee break while we run one more electrophoresis...
We have a nice visit today! Lauren, from the team of Pennsylvania, is here to tell us about her project, and learn a bit more about ours. Carlos is having an intense discussion on kinetics with her while Marta struggles to design the primers we'll need for the PCR reactions next week. There's no way to get them exactly right, what a nightmare!
Friday 22 July
It seems nothing's gone right so far except for the transformations. We'll have to start again...BUT IT ISN'T OVER 'TIL THE FAT LADY SINGS! [To be continued...]
Weekend
Blondie and little Rosidito didn't appear in the lab over the last two days so our bacterial cultures died and we have to waste time on Monday making new ones. The rest of the group are starting to hate both clumsy girls. [Just kidding, we love them: they're the best, so efficient and the most beautiful creatures]
