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YEAST PROTOCOLS
YEAST TRANSFORMATION
(Quick version for plasmid)
Preparation of solutions
1X TE/LiAc
Prepare fresh by using the 10X stock solutions provided with the kit. Dilutue in sterile dH2O
PEG/LiAc
Mix: 8 ml 50% PEG, 1 ml 10X TE, 10 ml 1M LiAc, all solutions provided with the kit
- Inoculate one or two colonies ( max 2 weeks old, 2-3 mm in diameter) into 100 ul 1X TE/LiAc-solution. Vortex
- Mix 0,2 ug plasmid with 100 ug herring testes carrier DNA (heat to ~100°C and cool before use) in an Eppendorf tube
- Add the resuspended cells and mix well
- Add 600 ul PEG/LiAc solution and vortex
- Incubate for 30 minutes at 30°C with shaking
- Add 70 ul DMSO and mix gently
- Heatshock for 15 minutes in a 42°C waterbath
- Plate the cells on selective media
YEAST DNA EXTRACTION PROTOCOL
(Lyticase protocol)
Lysis
- Inoculate 3 ml of SD/-Leu/-Trp/-His with one yeast colony and grow over night (or up to two days) at 30°C with shaking
- Spin down cells from 1,5 ml culture and resuspend in 500 ul lysis buffer
- Incubate at 37°C for 60 minutes
- Add 55 ul 10% SDS
- Add 3 ul 10 mg/ml Proteinase K
- Incubate at 56°C for 60 minutes
Extraction
- Add 600 ul Phenol:Chloroform:Isoamylalcohol (25:24:1), vortex
- Spin at 6000 rpm for 5 minutes
- Transfer the upper phase to a clean tube
Precipitation
- Add 1/10 vol of 3 M NaAc (pH 5,2) and 1 ml of 96% etanol
- Incubate at -80°C for 15 minutes
- Spin at 13 000 rpm for 10 minutes
- Resuspend in 20-30 ul of dH2O
Extraction of single plasmid
Transform E.coli (electroporation or chemically competent) with up to 1 ul of the yeast prep and use appropriate selection media for the plasmid you wish to extract
Lysis buffer
50 mM Tris-HCl pH 7,5
10 mM EDTA
0,5% b-mercaptoetanol
25 U Lyticase
4 ul RNase A
PCR-SCREENING OF POSITIVE COLONIES
PCR-reaction
| 10x advantage buffer |
2,5 ul |
| dNTP (2 mM) |
2,5 ul |
| 5'-primer |
2,0 pmol |
| 3'-primer |
2,0 pmol |
| Yeast DNA prep |
0,5 ul |
| Advantage 2 Polymerase |
0,5ul |
| dH2O to 25 ul |
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Cycling parameters
94°C for 1 min
30-35 cycles
94°C for 30 sek
68°C for 3 min
68°C for 3 min
soak at 15°C
Primers for pGADT7
Matchmaker 5' AD LD-Insert Screening Amplimer
CTA TTC GAT GAT GAA GAT ACC CCA CCAA ACC C
Matchmaker 3' AD LD-Insert Screening Amplimer
GTG AAC TTG CGG GGT TTT TCA GTA TCT ACG ATT
For addtional protocols, please consult Clontech's manuals
for Matchmaker Library Construction & Screening Kit, YEASTMAKER™ Transformation System 2 and Yeast Protocols Handbook.
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Last update 04.12.06 by Barbro E. Sæther
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