Team:Colombia/Journal

From 2014.igem.org

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==== '''15th June 2013''' ====
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We picked the transformant colonies.
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===='''18th June 2013'''====
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<p>We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.</p>
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<p>This are the overall steps:</p>
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<ol>
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<li>Harvest cells.</li>
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<li>Resuspend cells.</li>
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<li>Cell lysis.</li>
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<li>Neutralization.</li>
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Spin method:
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<li>Prepare column.</li>
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<li>Load cleared lysate.</li>
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<li>Wash column with wash solution 1.</li>
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<li>Was column with wash solution 2.</li>
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<li>Centrifuge.</li>
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<li>Elute DNA.</li>
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</ol>
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We did a confirmation Gel 100 V x 30 min. -> It showed 1 bond in the first two wells corresponding to Nal1 and Nal2. We succesfully extracted the Nal plasmids!
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===='''June 21st, 2013'''====
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Harju et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction
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<ol>
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<li>5 mL of overnight culture of ''S. cerevisiae'' (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.</li>
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<li>500 µL of Harju lysis buffer were added to each tube.</li>
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<li>Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.</li>
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<li>Vortex 30 s.</li>
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<li>Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.</li>
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<li>Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.</li>
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<li>Incubate for 5 min at room temperature or at 30 °C.</li>
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<li>Centrifuge for 5 min, 8500 rpm, and discard supernatant.</li>
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<li>Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.</li>
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<li>Dry pellets at room temperature or at 60 °C.</li>
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<li>Resuspend in 40 µL miliQ water.</li>
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</ol>
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===='''June 26, 2013'''====
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<ul>
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<li>We made competent yeast following the procedure mentioned before.</li>
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<li>We also made our first PCRs! We used primers 6 & 1 (A) and 34 & 9 (B) to extract VP16 and GCR from the Nal1 plasmid.</li>
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<li>Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
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<ol>
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<li>Ladder</li>
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<li>PCR A</li>
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<li>PCR B</li>
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<li>Miniprep for Nal. 1</li></ol></li>
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</ul>
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<p align="justify">
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A = VP16
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<br>B = GCR
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</p>
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[[File:Construct.jpg|400px|thumb|center]]
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===='''June 27, 2013'''====
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<p align="justify">
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We repeated the PCR for A and used lambda phage DNA for carrier DNA.
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We also tried extracting yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:
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<ul>
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<li>Method “H” used the regular lysis buffer.</li>
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<li>Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.</li>
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<li>Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.</li>
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</ul>
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</p>
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<p align="justify">
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Conformation gel was run with wells:
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<ol>
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<li>Ladder</li>
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<li>PCR A (repeated)</li>
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<li>Carrier lambda PCR</li>
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<li>Method C</li>
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<li>Method H</li>
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<li>Method O</li>
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</ol>
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The genome extraction still isn't working! :(
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</p>
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<p align="justify">
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We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:
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<ul>
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<li>1st PCR --> 2-step PCR
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<br>Cycle steps
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<ol>
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>15 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 5 min)</li>
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<li>Hold (4 °C, indefinite time)</li>
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</ol></li>
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<li>2nd PCR --> 2-step PCR (add primers)
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<br>Cycle steps
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<ol>
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>35 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 10 min)</li>
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<li>Hold (4 °C, indefinite time)</li>
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</ol></li>
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===='''July 2nd, 2013'''====
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Still trying to successfully extract the yeast genome! This time we tried an alternate method where we used two different solutions to break the cell wall:
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<ul>
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<li>Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized) + Zymolyase</li>
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<li>Solution II: NaOH 0.2 N, SPS 1%</li>
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The rest of the protocol was taken from GenElute DNA Kit from Sigma-Aldrich.
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</ul>
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===='''July 3rd, 2013'''====
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The genome extraction was better, but it's mostly degraded DNA! We still have to improve the protocol.
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===='''July 5th, 2013'''====
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We're still improving our genome extraction protocol. This time we're trying 4 variations to break the cell wall
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<br>We're varying the incubation of both solutions, the one that comes with the kit (Proteinase K + Lysis buffer) and the zymolyase solution we previously used. The four variations were as follows.
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<ul>
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<li>A: Zymolyase for ½ h at 37 °C</li>
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<li>B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C</li>
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<li>C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C</li>
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<li>D: Regular GenElute Genome extraction protocol</li>
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</ul>
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The rest of the steps were done following the instructions from the GenElute Genome extraction protocol.
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<br>We ran a gel in this order: WM, A, B, C, D.
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<br>Both B and C gave results, with C giving better yields! We're keeping the C protocol and we're happy we can start extracting parts from the yeast genome!
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[[File:GENOMES.jpg|400px|thumb|center]]
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<br>
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<br>We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
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<br>PCR fusion 2 steps*, with process 3 at 72 °C for 45 s
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<ol>*
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<li>98 °C, 1 min</li>
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<li>98 °C, 10 s</li>
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<li>72 °C, 45 s</li>
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Steps 2 and 3 x 35
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<li>72 °C, 10 min</li>
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<li>4 °C</li>
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</ol>
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===='''July 9th, 2013'''====
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Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!
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===='''July 18th, 2013'''====
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Today we ran PCRs for the following fusions: F1 = TER-GCR, with primers 31 & 35; F2 = TER – mCherry, with primers 31 & 33. We also amplified E = pGAL1, using primers 13 & 15.
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===='''July 19th, 2013'''====
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Today we amplified F2 that we obtained yesterday.
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===='''July 22nd, 2013'''====
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Today we tried to fuse C-D using primers 19 and 5, TER-GCR using primers 31 and 35 and TER-mCherry using primers 31 and 33. After 15 PCR cycles, we added the primers.
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===='''July 24th, 2013'''====
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Today we extracted the terminator from the yeast genome (F1 and F2) and pGAL1 from miniprep (E). We also did PCRs to fuse again our big parts: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19 & 34) and pGAL1 – mCherry (E-G) (primers 32 & 15).
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<br>At the moment, this is our progress!:
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[[File:Progress_chart.jpg|400px|thumb|center]]
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===='''August 5th, 2013'''====
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Things are advancing at a fast pace! Today we did miniprep to obtain PUC19!
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===='''August 6th, 2013'''====
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After doing a confirmation gel, C and CD are no longer with us! So we need to repeat them!
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===='''August 16th, 2013'''====
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Still no C nor D, so we did PCRs again with different methods to obtain C1, C2, D1 and D2.
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===='''August 17th,2013'''====
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We're now starting with the reporter construct's PCRs. We got the synthetised parts (E2, E3, E4 and F (the terminator)) which means we need to start fusing them. First off, we'll fuse the terminator with the reporter, since it's common to all reporters.
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===='''August 19th,2013'''====
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Today we'll perform the fusion between FG and the four Es.
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===='''August 20th,2013'''====
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The fusion didn't work out, so we reamplified FG and tried again.
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===='''August 30th, 2013'''====
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We finally have the E*GF fusions complete!
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We did the digestion for the E*GF parts and the pSB1C3 plasmid as follows:
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<ul>
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<li>Buffer 5x: 5 uL</li>
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<li>SpeI: 1 uL</li>
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<li>XbaI: 1 uL</li>
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<li>DNA: 10 uL </li>
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<li>Water: 33 uL </li>
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</ul>
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They were placed for two hours at 37°C and then were inactivated at 80°C for 20 minutes. Then we added alkaline phosphatase for 1 hour at 37°C.
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Then, we did the ligation protocol as follows:
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<ul>
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<li>Buffer 5x: 2 uL</li>
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<li>plasmid: 3 uL</li>
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<li>insert: 9 uL</li>
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<li>ligase: 1 uL</li>
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<li>Water: 5 uL</li>
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</ul>
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We then transformed them. Tomorrow we'll confirm the transformation by miniprep kit.
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===='''August 31st'''====
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After transforming them we got these minipreps
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[[File:GELminipreps.jpg|400px|thumb|center]]
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Here we can see all four E*GF parts (the four to the left of the weight marker), some with more than one plasmid conformation.
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===='''September 2nd, 2013'''====
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We contransformed the E*GF parts with the NAL plasmid in order to validate their function. Once again we did electrocompetent cells and transformed immediately.
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===='''September 3rd, 2013'''====
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We did the PCR confirmation for the parts. The bands aren't very bright but it's enough for us! We'll run the validation experiments tomorrow!
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Wells are as follows: E1FG - E1FG(2) - E2FG - E2FG(2) - Weight marker - E3FG - E3FG(2) - E4FG - E4FG(2).
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Something strange happened with E3FG(2)
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[[File:GEL7.jpg|400px|thumb|center]]
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===='''September 4th, 2013'''====
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Everything is ready for the validation experiments. We're both nervous and excited!
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We inoculated cells with dexamethasone. The original syringe had a concentration of 8mg/2mL. We obtained a total initial volume of 3.189*10^-2 µL, so we had to dilute first the dexamethasone: 9900 µL water + 100 µL dexamethasone.
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Induction experiment ON: 5 mL LB + 20 µL ampicillin + 20 µL kanamycin + 10 µL inoculum + 32 µL dexamethasone (1:1000) (inoculate ON).
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We ran a gel for several PCRs: F-H-P-E1GF2-MP-E2GF-E3GF1-E3GF2-E4GF- Everything was succesful except E1GF2.
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Then we digested the PCRs with 5 µL Buffer CutSmart + 15 µL PCR + 1 µL XbaI + 1 µL SpeI + 28 µL water (for each PCR)
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<html></p>
<html></p>

Revision as of 16:58, 19 June 2014

Journal

CONTENIDO

Lalalala.

CONTENIDO 2

Lalalalla

  1. ej1
  2. ej2
  3. ej3


COTENIDO OTRO MES

Lalalala

Retrieved from "http://2014.igem.org/Team:Colombia/Journal"