"
Team:Goettingen/protocol Colony
From 2014.igem.org
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.
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.
