Team:Colombia Uniandes/Protocols
From 2013.igem.org
Contents
Protocol for extraction of yeast genome:
GenElute DNA Kit from Sigma-Aldrich with modifications
- 1,5 mL 2 min x 15000 rpm.
- 100 µL zymolyase solution.
- Vortex.
- Incubate 37°C x 30 min.
- 180 µL of Lysis Solution T.
- 20 µL of Proteinase K.
- Incubate 37°C x 30 min.
- 200 µL of Lysis Solution C.
- Vortex x 15 s.
- Incubate 55°C x 10 min.
- Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
- 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.
- 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.
- 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- The final eluate contains the DNA – Do not discard!
Protocol for electrocompetent cells
- Divide the ON culture in 50 mL falcon tubes.
- Centrifuge 8000 rpm x 10 min.
- Discard supernatant.
- Resuspend everything with water in two falcons.
- Centrifuge again.
- Discard supernatant and resuspend with water. Wash with water two more times.
- Centrifuge again.
- Discard supernatant.
- Resuspend with glycerol with water. Glycerol 10%.
- Centrifugue.
- Repeat steps 8-10.
- 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
- Add 2-3 colonies from a dish to 1 mL of medium.
- Vortex.
- Transfer the milliliter to 50 mL of medium.
- Incubate: 30 °C, 250 rpm, 16-18 h.
- When OD = 0.2-0.3, transfer 30 mL to 300 mL of medium.
- Incubate: 30 °C, 230 rpm, 3 h.
- When OD = 0.4-0.6, preferably 0.6, redistribute the medium with yeast cells among 50 mL falcons.
- Centrifuge: 1000 g, 5 min, 20 °C.
- Discard supernatant and resuspend with water or TE (the pellet ends up floating).
- Mix all the contents of all the falcons in another falcon, achieving total volume of 25-50 mL.
- Centrifuge 1000 g, 5 min, 20-25 °C.
- Discard supernatant.
- Add 1.5 mL of 1xTE/1xLiAc solution to the pellet. This cells are now competent.
Transformation:
- In a 1.5 mL tube, mix 0.1 µg of the plasmid and 0.1 mg of carrier.
- Mix with 0.1 mL of competent cells.
- Vortex.
- Add 0.6 mL of PEG/LiAc solution and vortex.
- Incubate: 30 °C, 30 min, 200 rpm.
- Add 70 µL of DMSO and mix by inverting. Do not vortex.
- Put the tube in water bath at 42 °C for 15 min. (Thermic shock).
- Cool on ice for 1-2 min.
- Centrifuge 14000 rpm, 5 s, 20-25 °C.
- Discard supernatant.
- Resuspend with 0.5 mL of 1x TE.
- Spread 100 µL in a Petri dish using pearls.
- 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.
Contents |
Protocol for extraction of yeast genome:
GenElute DNA Kit from Sigma-Aldrich with modifications
- 1,5 mL 2 min x 15000 rpm.
- 100 µL zymolyase solution.
- Vortex.
- Incubate 37°C x 30 min.
- 180 µL of Lysis Solution T.
- 20 µL of Proteinase K.
- Incubate 37°C x 30 min.
- 200 µL of Lysis Solution C.
- Vortex x 15 s.
- Incubate 55°C x 10 min.
- Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
- 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.
- 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.
- 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- The final eluate contains the DNA – Do not discard!
Protocol for electrocompetent cells
- Divide the ON culture in 50 mL falcon tubes.
- Centrifuge 8000 rpm x 10 min.
- Discard supernatant.
- Resuspend everything with water in two falcons.
- Centrifuge again.
- Discard supernatant and resuspend with water. Wash with water two more times.
- Centrifuge again.
- Discard supernatant.
- Resuspend with glycerol with water. Glycerol 10%.
- Centrifugue.
- Repeat steps 8-10.
- 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
- Add 2-3 colonies from a dish to 1 mL of medium.
- Vortex.
- Transfer the milliliter to 50 mL of medium.
- Incubate: 30 °C, 250 rpm, 16-18 h.
- When OD = 0.2-0.3, transfer 30 mL to 300 mL of medium.
- Incubate: 30 °C, 230 rpm, 3 h.
- When OD = 0.4-0.6, preferably 0.6, redistribute the medium with yeast cells among 50 mL falcons.
- Centrifuge: 1000 g, 5 min, 20 °C.
- Discard supernatant and resuspend with water or TE (the pellet ends up floating).
- Mix all the contents of all the falcons in another falcon, achieving total volume of 25-50 mL.
- Centrifuge 1000 g, 5 min, 20-25 °C.
- Discard supernatant.
- Add 1.5 mL of 1xTE/1xLiAc solution to the pellet. This cells are now competent.
Transformation:
- In a 1.5 mL tube, mix 0.1 µg of the plasmid and 0.1 mg of carrier.
- Mix with 0.1 mL of competent cells.
- Vortex.
- Add 0.6 mL of PEG/LiAc solution and vortex.
- Incubate: 30 °C, 30 min, 200 rpm.
- Add 70 µL of DMSO and mix by inverting. Do not vortex.
- Put the tube in water bath at 42 °C for 15 min. (Thermic shock).
- Cool on ice for 1-2 min.
- Centrifuge 14000 rpm, 5 s, 20-25 °C.
- Discard supernatant.
- Resuspend with 0.5 mL of 1x TE.
- Spread 100 µL in a Petri dish using pearls.
- 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.