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.

Test for auto activity of bait constructs

Background: The constructed strains carring the bait plasmid should not grow on plates without histidine, the promoter for histidine should be active only after mating and positive interaction. Sometimes the gene is transcribed and false positives colonies occur.

1. Plating the yeast strains transformed with the bait construct onto SC double drop-out plates lacking Tryptophane and Histidine and supplemented with 3-AT (6 mM) to suppress the background leakage of the Gal4-dependent promoters. Streak out the same strain on plates without Trp, to save the colonies.
2. No growth should occur on the plates without Histidine. Then the colonies on plates containing Histidine can be used for further experiments
3. In case of growth on these control plates the promoter activity is too high and colonies cannot be used for the Yeast-Two-Hybrid experiment.

Yeast two hybrid assay with robot

1. Inoculate desired strains (bait and prey) in selection medium over night.
2. Pipette 200 µl of each strain into a 96-well plate for robot screen or measure OD600 and spot same cell amount onto SC plates. Let this grow over night.
3. Use mating program of the robot, where it picksand transfers a colony to a mating plate or stamp colonies from SC plates to YPAD plate.
4. Mated colonies should be transferred to SC plates without aselection marker to select only for interactions, supplementation with 6mM 3-AT avoids auto-induction. Grown colonies indicate an interaction.
5. Transfer same cell amount with the robot and spot cells after OD600 measurement onto plates without marker, 3-AT and X-α-Gal. Growth over night and check for blue colonies.