Team:Colombia Uniandes/ChimiJournal

=='''Dear Journal! :)'''== 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. ===='''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'''====

• Competent yeast were made following the procedure mentioned before.
• PCR using primers 6 & 1 (A) and 34 & 9 (B) was run to extract VP16 from the Nal1 plasmid and GCR.
• 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