Team:Colombia Uniandes/ChimiJournal

== '''13th June 2013''' ==

Take out plasmid 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.

Electroporation: Use an ice tray with ice to take out the competent cells. The cells are in the Biophysics revco, the one shared with the LAMFU on the second floor of the J building. The revco is opened with the keys of the Biophysics keyring and the cells are on the top right drawer, in the middle. (Look at figure 1) == '''15th June 2013''' == The transformant colonies were stung (César, Alejandro & Silvia). =='''18th June 2013'''==

Procedure: Miniprep with GenElute HP Plasmid Miniprep kit.

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.

• Miniprep with GenElute HP Plasmid Miniprep Kit.
• Confirmation Gel 100 V x 30 min. -> 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