Team:Colombia Uniandes/Protocols

Contents 1 Instructions for gel preparation: 2 Instructions for LB medium preparation 2.1 LB liquid medium preparation (1 L) 2.2 LB solid medium preparation (1 L) 3 Protocol for extraction of yeast genome: 3.1 GenElute DNA Kit from Sigma-Aldrich with modifications 4 Protocol for electrocompetent cells 5 Transformation of yeast 6 Chemocompetent cells: 7 Genome Extraction:

Instructions for gel preparation:

1. Weigh 0,3g of agarose.
2. Add 30 mL of TAE 1x.
3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
5. When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
6. Pour the solution into the bed and clear all its bubbles with a tip. Put a piece of paper on top of it and let it polymerize.
7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.

Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.

Instructions for LB medium preparation

LB liquid medium preparation (1 L)

• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water

LB solid medium preparation (1 L)

• 15 g agar agar
• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water

Protocol for extraction of yeast genome:

GenElute DNA Kit from Sigma-Aldrich with modifications

1. 1,5 mL 2 min x 15000 rpm.
2. 100 µL zymolyase solution.
3. Vortex.
4. Incubate 37°C x 30 min.
5. 180 µL of Lysis Solution T.
6. 20 µL of Proteinase K.
7. Incubate 37°C x 30 min.
8. 200 µL of Lysis Solution C.
9. Vortex x 15 s.
10. Incubate 55°C x 10 min.
11. Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
12. 200 µL of ethanol (100%) to the lysate – Vortex. --> Transfer the entire contents of the tube into the binding column --> 12000 rpm x 1 min --> Discard the collection tube and place the column in a new one.
13. 500 µL of Wash Solution 1 to the column. --> 12000 rpm x 1 min --> Discard the collection tube and place the column in a new one.
14. 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
15. 30 µL of water (miliQ) --> 14000 rpm x 1 min.
16. 30 µL of water (miliQ) --> 14000 rpm x 1 min.
17. The final eluate contains the DNA – Do not discard!

Protocol for electrocompetent cells

1. Divide the ON culture in 50 mL falcon tubes.
2. Centrifuge 8000 rpm x 10 min.
3. Discard supernatant.
4. Resuspend everything with water in two falcons.
5. Centrifuge again.
6. Discard supernatant and resuspend with water. Wash with water two more times.
7. Centrifuge again.
8. Discard supernatant.
9. Resuspend with glycerol with water. Glycerol 10%.
10. Centrifugue.
11. Repeat steps 8-10.
12. Discard supernatant and divide what is left in eppendorfs.

CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).

Transformation of yeast

1. Add 2-3 colonies from a dish to 1 mL of medium.
2. Vortex.
3. Transfer the milliliter to 50 mL of medium.
4. Incubate: 30 °C, 250 rpm, 16-18 h.
5. When OD = 0.2-0.3, transfer 30 mL to 300 mL of medium.
6. Incubate: 30 °C, 230 rpm, 3 h.
7. When OD = 0.4-0.6, preferably 0.6, redistribute the medium with yeast cells among 50 mL falcons.
8. Centrifuge: 1000 g, 5 min, 20 °C.
9. Discard supernatant and resuspend with water or TE (the pellet ends up floating).
10. Mix all the contents of all the falcons in another falcon, achieving total volume of 25-50 mL.
11. Centrifuge 1000 g, 5 min, 20-25 °C.
12. Discard supernatant.
13. Add 1.5 mL of 1xTE/1xLiAc solution to the pellet. This cells are now competent.

Transformation:

1. In a 1.5 mL tube, mix 0.1 µg of the plasmid and 0.1 mg of carrier.
2. Mix with 0.1 mL of competent cells.
3. Vortex.
4. Add 0.6 mL of PEG/LiAc solution and vortex.
5. Incubate: 30 °C, 30 min, 200 rpm.
6. Add 70 µL of DMSO and mix by inverting. Do not vortex.
7. Put the tube in water bath at 42 °C for 15 min. (Thermic shock).
8. Cool on ice for 1-2 min.
9. Centrifuge 14000 rpm, 5 s, 20-25 °C.
10. Discard supernatant.
11. Resuspend with 0.5 mL of 1x TE.
12. Spread 100 µL in a Petri dish using pearls.
13. Incubate: 30 °C, 2-4 days.

Solutions:

• 10x LiAc: 1 M lithium acetate, pH 7.5 with acetic acid. Autoclave.
• 10x TE: 0.1 M Tris HCl, 10 mM EDTA, pH 7.5. Autoclave.
• PEG/LiAc 10 mL: 8 mL PEG 50%, 1 mL TE 10x, 1 mL 10x LiAc.

Chemocompetent cells:

We used TransformAID Bacterial Transformation Kit, Thermo Scientific according to manufacturer's instructions. You can review the protocol in the following link: File:TransformAid Bacterial Transformation Kit.pdf.

Genome Extraction:

For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to manufacturer's instructions:File:Easy-DNA Kit.pdf