Team:Goettingen/protocol Colony

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         <h2 id="E_cra"><i>E.coli</i> cracking</h2>
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         <h3 id="E_cra"><i>E.coli</i> cracking</h3>
             <p>Solutions: <br />
             <p>Solutions: <br />
             &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Crack buffer: 100 µl 2 M NaOH (0.16 g/ 2 ml), 50 µl 10% SDS, 0,2 g Glc, add 1 ml H2O<br />
             &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Crack buffer: 100 µl 2 M NaOH (0.16 g/ 2 ml), 50 µl 10% SDS, 0,2 g Glc, add 1 ml H2O<br />
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             8. Under UV-illuminator, plasmid DNA should be visible between <i>E. coli</i> genomic DNA (20-30 kb) and low molecular weight RNAs.<br /><br />
             8. Under UV-illuminator, plasmid DNA should be visible between <i>E. coli</i> genomic DNA (20-30 kb) and low molecular weight RNAs.<br /><br />
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             </p>
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             <h2 id="Y_col">Yeast colony PCR</h2>
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             <p>1. Aliquot 20 µl NaOH (20 mM) into 1.5 ml tubes <br />
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            2. Pick colonies (use pipet tips) into the NaOH <br />
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             <h3 id="Auto_act">Test for auto activity of bait constructs</h3>
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            3. Incubate at 95°C for ~ 45 minutes <br />
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             <p>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. <br /><br />
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            4. Centrifuge at max speed for 10 minutes <br />
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             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.<br />
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             5. Use 5 µl of supernatant as template in a (50 µl) PCR. <br /><br />
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2. No growth should occur on the plates without Histidine. Then the colonies on plates containing Histidine can be used for further experiments<br />
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            </p>
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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. <br /><br />
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            <h2 id="Auto_act">Auto-activity Test</h2>
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            <p>plating the yeast strains transformed with the bait construct onto SC double drop-out plates lacking Trp and His<br />
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            supplemented with 3-AT (3-5 mM) to suppress the background leakage of the Gal4-dependent promoters<br />
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            in case of growth on these control plates, the 3-AT concentration can be increased up to 50-100 mM<br /><br />
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</p>
</p>
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            <h2 id="Y2H_Scr">Yeast-Two-Hybrid Screening</h2>
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            <p>
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<h3 id="Y2H_robot">Yeast two hybrid assay with robot</h3>
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            1. Inoculate bait strain in 50 ml SC-Trp + 4% Glc <br />
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<p>
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            2. Incubate O/N at 30°C, 200 rpm <br />
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1. Inoculate desired strains (bait and prey) in selection medium over night.<br />
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            3. Thaw library aliquot into 42°C water bath <br />
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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.<br />
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            4. Resuspend library in YPAD (~55 ml/screening) OD 600= 0.1 per ml<br />
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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.<br />
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            5. Incubate at least 1 h at 30°C, 200 rpm<br />
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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.<br />
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            6. Determine the OD 600 of bait and prey<br />
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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. <br /><br />
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            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)<br />
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</p>
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            8. Discart supernatant, resuspend pellet in 10 ml YPAD + 20% PEG 6000 and transfer to 100 ml flask<br />
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             <p>Watch the following tutorial to learn about the whole process:<br /></p>
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            9. Incubate 4-5 h (or O/N) at 30°C only 80 rpm<br />
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<center><video width="640" height="390" controls><source src="https://static.igem.org/mediawiki/2014/2/2a/Goettingen2014-New_Robot.mp4" type="video/mp4">Your browser does not support the video tag.</video></center><br /><br />
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             10. Centrifuge for 4 min at 4000 rpm<br />
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            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<br />
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<h3 id="Y2H_screen">Yeast two hybrid Screening</h3>
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            12. Distribute the mix on 10-15 plates incubate 5-10 days at 30°C<br />
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             <p>Watch the following tutorial to learn about the whole process:<br /></p>
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            13. Transfer 10 µl of a grown colony to 100 µl SD-Leu+Trp+His medium (96 well)<br />
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             <center><video width="640" height="390" controls><source src="https://static.igem.org/mediawiki/2014/5/5a/Goettingen2014-Y2H_Screening.mp4" type="video/mp4">Your browser does not support the video tag.</video> </center>
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            14. Transfer to SC-Leu+Trp+His agar plate<br />
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            15. Analyze preys by yeast colony PCR and sequencing <br /><br />        
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            </p>
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             <br /><br />
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            <h2 id="Y2H_bw">Y2H part II - blue/white selection</h2>
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             <p>
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<div><a href="https://2014.igem.org/Team:Goettingen/protocol_Plasmid_Tran" class="button_pre"><b>Previous</b></a><a href="https://2014.igem.org/Team:Goettingen/protocol_protein" class="button_next"><b>Next</b></a></div>
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            1. Pick colonies to 96 well plate into 50 µl H2O with sterile tip<br />
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            2. Plate on LWH plates, with diff. Concentrations of 3-AT <br />
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             3. Plasmid isolation and re-Transformation<br />
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            4. Blue/ White screen<br /><br />
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            Material:<br />
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            &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Glass beads (425-600 µm)<br />
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            &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Resuspension Buffer (P1): 50 mM Tris.Cl, pH 8.0, 10 mM EDTA, 100 µg/ml RNase A<br />
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            &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Lysis buffer (P2): 200 mM Na OH, 1% SDS (w/v)<br />
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            &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Neutralization buffer (N3): 3.0 M potassium acetate pH 5.5<br /><br />
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            1. Pick a yeast colony into 5 ml of triple drop-out media. Incubate overnight (20h) at 30°C with shaking (250rpm)<br />
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            or: Collect (wash off) well-grown yeast colonies directly from the plates for ‘pool’ preparation (10-25 OD 600 units).<br />
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            2. Collect cells by centrifugation (room temperature, 4000 rpm, 5 min) and discard the supernatant<br />
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            3. Resuspend the pellet in 250 µl of buffer P1.<br />
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             4. Add ca. 0.4 g glass beads and break the cells by vortexing vigorously for 5 min.<br />
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            5. Add 250 µl of buffer P2 and mix immediately<br />
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            6. Add 350 µl of buffer N3 and mix throughly<br />
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            7. Purify plasmid (phenol/chloroform extraction and ethanol precipitation).<br /><br />
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            <b>blue/white selection:</b><br />
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            8. Transform <i>E.coli</i> 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.<br />
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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 />
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            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 />
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            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).<br />
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            12. Analyze inserts from the prey plasmids of specifically interacting candidates by colony PCR and sequencing. <br /><br />
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            </p>
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         </div>
         </div>
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Latest revision as of 21:12, 17 October 2014

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.

Watch the following tutorial to learn about the whole process:



Yeast two hybrid Screening

Watch the following tutorial to learn about the whole process:




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