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Team:Goettingen/protocol Colony
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9. Isolate the plasmid from <i>E.coli</i> by conventional plasmid preparation methods ‘Pool’ preparation: wash <i>E.coli</i> colonies off the plates, combine and prepare plasmid from the mix.<br /> | 9. Isolate the plasmid from <i>E.coli</i> by conventional plasmid preparation methods ‘Pool’ preparation: wash <i>E.coli</i> colonies off the plates, combine and prepare plasmid from the mix.<br /> | ||
10. Transform MATalpha, Y187 with the isolated prey plasmid (pool), select on media lacking Leucine (SC-L). The number of transformants should exceed the number of positive candidates obtained from the screening.<br /> | 10. Transform MATalpha, Y187 with the isolated prey plasmid (pool), select on media lacking Leucine (SC-L). The number of transformants should exceed the number of positive candidates obtained from the screening.<br /> | ||
| - | 11. | + | 11. Automation step: Prepare 96(384)-well plates one with AlG0X-strain and the other with YIG0X-strain. Individually mate each prey-containing Y-strain with either the original bait-containing AlG-strain, also use control bait (e.g. GFP). Select for zygote formation in well-plate (SD-LW) and for interaction (SD-LWH + 3-AT + x-GAL).<br /> |
12. Analyze inserts from the prey plasmids of specifically interacting candidates by colony PCR and sequencing. <br /><br /> | 12. Analyze inserts from the prey plasmids of specifically interacting candidates by colony PCR and sequencing. <br /><br /> | ||
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Revision as of 11:05, 20 September 2014
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
E.coli cracking
Solutions:
Crack buffer: 100 µl 2 M NaOH (0.16 g/ 2 ml), 50 µl 10% SDS, 0,2 g Glc, add 1 ml H2O
Crack dye: 150 µl 4 M KCl (0.597 g / 2 ml), 50 µl Loading-dye
1. Pick bacterial colonies to a large size (2-3 mm)
2. Using a sterile tip, transfer a small quantity of the colony to 1.5 ml cup containing 10 µL 10 mM EDTA add 25 µl Crack buffer
3. Incubate the tube at 70 °C 5 minutes
4. Cool down on ice.
5. Add 2 µl Crack dye and incubate 10 min on ice
6. Spin down 10 minutes 14K rpm
7. Run a gel with 25 µl of the supernatant. (Note: it is difficult to apply the mixture because of its viscosity. Loading the mixtures into empty wells rather than the wells filled with buffer and pouring buffer thereafter may give better result.)
8. Under UV-illuminator, plasmid DNA should be visible between E. coli genomic DNA (20-30 kb) and low molecular weight RNAs.
Yeast colony PCR
1. Aliquot 20 µl NaOH (20 mM) into 1.5 ml tubes
2. Pick colonies (use pipet tips) into the NaOH
3. Incubate at 95°C for ~ 45 minutes
4. Centrifuge at max speed for 10 minutes
5. Use 5 µl of supernatant as template in a (50 µl) PCR.
Auto-activity Test
plating the yeast strains transformed with the bait construct onto SC double drop-out plates lacking Trp and His
supplemented with 3-AT (3-5 mM) to suppress the background leakage of the Gal4-dependent promoters
in case of growth on these control plates, the 3-AT concentration can be increased up to 50-100 mM
