Team:Goettingen/protocol Colony

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

Yeast-Two-Hybrid Screening

1. Inoculate bait strain in 50 ml SC-Trp + 4% Glc
2. Incubate O/N at 30°C, 200 rpm
3. Thaw library aliquot into 42°C water bath
4. Resuspend library in YPAD (~55 ml/screening) OD 600= 0.1 per ml
5. Incubate at least 1 h at 30°C, 200 rpm
6. Determine the OD 600 of bait and prey
7. Mix 10 OD bait with 10 OD prey (ex: OD bait= 3.5/ OD prey= 2 , 10OD bait=2.8 ml/ 10OD prey= 5 ml), soin down (4 min, 4 krpm, RT)
8. Discart supernatant, resuspend pellet in 10 ml YPAD + 20% PEG 6000 and transfer to 100 ml flask
9. Incubate 4-5 h (or O/N) at 30°C only 80 rpm
10. Centrifuge for 4 min at 4000 rpm
11. Discart supernatant, resuspend in 2ml SC-Leu+Trp+His, add to semi-solid medium, mix by shaking titer plates: 10 20 50 µl spred on SD-Leu+Trp + Glc incubate 2 days at 30°C, count colonies, calc. mating efficiency
12. Distribute the mix on 10-15 plates incubate 5-10 days at 30°C
13. Transfer 10 µl of a grown colony to 100 µl SD-Leu+Trp+His medium (96 well)
14. Transfer to SC-Leu+Trp+His agar plate
15. Analyze preys by yeast colony PCR and sequencing

Y2H part II - blue/white selection

1. Pick colonies to 96 well plate into 50 µl H2O with sterile tip
2. Plate on LWH plates, with diff. Concentrations of 3-AT
3. Plasmid isolation and re-Transformation
4. Blue/ White screen

Material:
     Glass beads (425-600 µm)
     Resuspension Buffer (P1): 50 mM Tris.Cl, pH 8.0, 10 mM EDTA, 100 µg/ml RNase A
     Lysis buffer (P2): 200 mM Na OH, 1% SDS (w/v)
     Neutralization buffer (N3): 3.0 M potassium acetate pH 5.5

1. Pick a yeast colony into 5 ml of triple drop-out media. Incubate overnight (20h) at 30°C with shaking (250rpm)
or: Collect (wash off) well-grown yeast colonies directly from the plates for ‘pool’ preparation (10-25 OD 600 units).
2. Collect cells by centrifugation (room temperature, 4000 rpm, 5 min) and discard the supernatant
3. Resuspend the pellet in 250 µl of buffer P1.
4. Add ca. 0.4 g glass beads and break the cells by vortexing vigorously for 5 min.
5. Add 250 µl of buffer P2 and mix immediately
6. Add 350 µl of buffer N3 and mix throughly
7. Purify plasmid (phenol/chloroform extraction and ethanol precipitation).

blue/white selection:
8. Transform E.coli and select transformants on the appropriate dropout media to select for the prey plasmid. The number of transformants should exceed the number of positive candidates obtained from the screening.
9. Isolate the plasmid from E.coli by conventional plasmid preparation methods ‘Pool’ preparation: wash E.coli colonies off the plates, combine and prepare plasmid from the mix.
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.
11. Robot: 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).
12. Analyze inserts from the prey plasmids of specifically interacting candidates by colony PCR and sequencing.