Team:Colombia Uniandes/ChimiJournal

Hands at work!

Here you will find the overall progression of our work at the laboratory designing Chimi.

13th June 2013

The first thing we did was to extract the plasmids from the iGEM plaque.

1. From the 2013 kit, Plate 1, Well 19 – o
2. From the 2013 kit, Plate 1, Well 2 – i
3. From the 2013 kit, Plate 3, Well 17 – c

The iGEM parts were taken in order to perform an electroporation. For this, we use:

• 20 µ of miliQ water (ultra pure). Find the plate and stick the tip with water into the well, perforating the aluminum.
• Resuspend the well’s content by gentle pipetting.
• When the water has a dark red color, transfer it to a PCR eppendorf and put on ice.

15th June 2013

We stung the transformant colonies.

18th June 2013

We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.

This are the overall steps:

1. Harvest cells.
2. Resuspend cells.
3. Cell lysis.
4. Neutralization.

Spin method:

5. Prepare column.
6. Load cleared lysate.
7. Wash column with wash solution 1.
8. Was column with wash solution 2.
9. Centrifuge.
10. Elute DNA.

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!

June 21, 2013

Harja et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction

1. 5 mL of overnight culture of S. cerevisiae (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.
2. 500 µL of Harja lysis buffer were added to each tube.
3. Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.
4. Vortex 30 s.
5. Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.
6. Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.
7. Incubate for 5 min at room temperature or at 30 °C.
8. Centrifuge for 5 min, 8500 rpm, and discard supernatant.
9. Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.
10. Dry pellets at room temperature or at 60 °C.
11. Resuspend in 40 µL miliQ water.

June 26, 2013

• We made competent yeast following the procedure mentioned before.
• 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.
• Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
  1. Ladder
  2. PCR A
  3. PCR B
  4. Miniprep for Nal. 1

A = VP16
B = GCR

June 27, 2013

We repeated the PCR for A and used lambda phage DNA for carrier DNA. We also extracted yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:

• Method “H” used the regular lysis buffer.
• Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.
• Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.

Conformation gel was run with wells:

1. Ladder
2. PCR A (repeated)
3. Carrier lambda PCR
4. Method C
5. Method H
6. Method O

We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:

• 1st PCR --> 2-step PCR
  Cycle steps
  1. Initial denaturation (98 °C, 30 s)
  2. 15 cycles (98 °C, 10 s; 72 °C, 35 s)
  3. Final extension (72 °C, 5 min)
  4. Hold (4 °C, indefinite time)
• 2nd PCR --> 2-step PCR (add primers)
  Cycle steps
  1. Initial denaturation (98 °C, 30 s)
  2. 35 cycles (98 °C, 10 s; 72 °C, 35 s)
  3. Final extension (72 °C, 10 min)
  4. Hold (4 °C, indefinite time)

June 28th, 2013

We extracted pBap2, Gal4 and pGal1 (C, D and E) from the genome of S. cerevisiae. We ran a gel with the following wells: WM, AB, C, D, E.

July 2nd, 2013

We ran out of yeast genome, so today we had to extract it again! We used two different solutions:

• Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized)
• Solution II: NaOH 0.2 N, SPS 1%

July 5th, 2013

We performed yeast genome extraction using 4 methods.
General guidelines from the GenElute DNA Kit from Sigma-Aldrich.

• A: Zymolyase for ½ h at 37 °C
• B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C
• C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C
• D: Normal protocol


We ran a gel in this order: WM, A, B, C, D. (Positive for B, A, C. Brighter band in C.)
We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
PCR fusion 2 steps*, with process 3 at 72 °C for 45 s

*
1. 98 °C, 1 min
2. 98 °C, 10 s
3. 72 °C, 45 s

Steps 2 and 3 x 35

4. 72 °C, 10 min
5. 4 °C

July 9th, 2013

Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!

<il>pBap2 = C
• GAL4 = D
• VP16 = A
• GCR = B
• TER = F
• VP16 + GCR = AB
• pBAP2 + GAL4 = CD
• AB + TER = ABF

July 18th PCR (F1 = TER-GCR, primers 31&35) (F2 = TER – mCherry, primers 31&33) (E = pGAL1, primers 13&15) 12.5 µL Master Mix 1.25 µL FW 1.25 µL RV 2.5 µL DNA (F1 & F2: 1.5µL DNA) 1.25 µL DMSO 6.25 µL wáter TOTAL: 25 µL July 19th Amplification of F2 (july 18th) 12.5 µL Master Mix 1 µL each primer (31&33) 5 µL template (F2) 1.25 µL DMSO 5.25 µL water Total: 25 µL July 22nd PCR for C-D fusión (primers 19 and 5) and TER-GCR (primers 31 and 35). TER-mCherry (31 and 33) July 24th Terminator extraction from yeast genome (F1 and F2) pGAL1 extraction from miniprep (E) PCRs: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19&34)

pGAL1 – mCherry (E-G) (primers 32-15)