Team:MIT phageprot

From 2010.igem.org

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<li><a href="https://2010.igem.org/Team:MIT_phageprot">Basic Protocol</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phageprot">Experimental Protocols</a></li>
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<td><div class="bodybaby">TITLE</div></td>
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<td><div class="bodybaby">Phage Experimental Methods</div></td>
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YOUR TEXT HERE.
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<b>Large-scale M13 Amplification</b><br>
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To produce single-M13 phage that incorporate our fusion protein into the phage coat, we first transform XL1-Blue cells with a plasmid with our fusion. Colonies are picked from the plated transformation,  grown overnight at 37C in 10 mL LB with selection. The overnight culture is diluted 1:100 in 1L LB with selection, and grown at 37C to mid-log phase. When the cells have reached mid-log phase, M13K07 helper phage is added to a concentration of approximately 10 phage per cell. Since the pLux/CI promoter that we used is leaky, we do not induce. The infected cells are left to amplify at 37C for 5 hours. After this time, the culture is ready for purification.
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<br><br>
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<b>M13 Phage Purification</b><br>
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Cells are spun down at 8,000 g for 30 minutes. The supernatant is carefully transferred to fresh tubes,  and re-spun. The top 80% of the supernatant is transferred to a clean tube. One-sixth volume of 20% PEG-8000 / 2.5M NaCl is added to the tubes. The phage are precipitated overnight at 4C, then spun at 8,000g for 30 minutes. The supernatant is discarded, and the pellet is resuspended in 5mL TBS. The supernatant is spun to remove residual bacteria, and then PEG precipitated again (45min-overnight at 4C). After spinning and decanting the supernatant, the phage are resuspended in TBS, and spun again to remove bacteria. This is the final phage stock. The concentration can be determined by titer and nanodrop.
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<br><br>
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<b>M13 Titer</b><br>
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Melted LB top agar is placed into 3mL aliquots and kept at 47C. A series of sterile tubes are labeled according to the dilution factor. 100 ul of E. coli strain ER2738 overnight culture are added to each of these tubes. Desired serial dilutions are prepared. 10 ul of each dilution are placed in the corresponding culture aliquot, and mixed gently. One aliquot of cells is transferred to an aliquot of top agar, then poured into a small petri dish with no bottom agar. This step is repeated for each aliquot of cells. The top agar is allowed to solidify, then transferred to the 37 degree incubator. Plates are checked for plaques after 8-12 hours.
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<br><br>
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<b>Nanodropping M13 Phage</b><br>
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Purified phage solutions can be nanodropped to obtain an estimate of the particles/mL. The 269 nm and the 320 nm absorbances are measured, and plugged into the following equations:
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<img src ="https://static.igem.org/mediawiki/2010/4/4d/Methodseqns.nb_1.gif"><br>
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<img src ="https://static.igem.org/mediawiki/2010/f/f4/Methodseqns.nb_2.gif">
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<br><br>
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<b>SDS-Page of Phage</b><br>
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Phage sample is treated with DNAse and left to incubate for 10 minutes at room temperature. 3x SDS sample buffer with BME is added, then boiled for 10 minutes. Samples are loaded into 14% SDS gel. The gel is run for 50 minutes at 170 V.
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<br><br>
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<b>Western Blot</b><br>
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A PVDF membrane is soaked in methanol briefly. BioRad Criterion blotter is assembled according to the manual with the gel and the membrane. The blot is run overnight at 20V at 4C with an ice block and a magnetic stirbar. After transfer, the membrane is placed in blocking buffer (1% BSA in 1X TBST) for an hour. The membrane is then washed three times for five minutes in TBST. A primary antibody solution is prepared in .3% BSA in TBST. The membrane is placed in the antibody solution for 1 hour, then washed three times. Probing with the secondary occurs in the same manner. After the wash sequence, the colorimetric substrate solution is added. When bands appear, the reaction is stopped by placing the membrane in water.
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<br><br>
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<b>AFM Sample Prep</b><br>
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70ul of culture is applied to a freshly cleaved mica surface. The sample is left to sit for 20 minutes, then dipped in Milli-Q water for several seconds to wash. After being dried with nitrogen gas, the sample is imaged.

Revision as of 17:54, 27 October 2010

Phage Experimental Methods
Large-scale M13 Amplification
To produce single-M13 phage that incorporate our fusion protein into the phage coat, we first transform XL1-Blue cells with a plasmid with our fusion. Colonies are picked from the plated transformation, grown overnight at 37C in 10 mL LB with selection. The overnight culture is diluted 1:100 in 1L LB with selection, and grown at 37C to mid-log phase. When the cells have reached mid-log phase, M13K07 helper phage is added to a concentration of approximately 10 phage per cell. Since the pLux/CI promoter that we used is leaky, we do not induce. The infected cells are left to amplify at 37C for 5 hours. After this time, the culture is ready for purification.

M13 Phage Purification
Cells are spun down at 8,000 g for 30 minutes. The supernatant is carefully transferred to fresh tubes, and re-spun. The top 80% of the supernatant is transferred to a clean tube. One-sixth volume of 20% PEG-8000 / 2.5M NaCl is added to the tubes. The phage are precipitated overnight at 4C, then spun at 8,000g for 30 minutes. The supernatant is discarded, and the pellet is resuspended in 5mL TBS. The supernatant is spun to remove residual bacteria, and then PEG precipitated again (45min-overnight at 4C). After spinning and decanting the supernatant, the phage are resuspended in TBS, and spun again to remove bacteria. This is the final phage stock. The concentration can be determined by titer and nanodrop.

M13 Titer
Melted LB top agar is placed into 3mL aliquots and kept at 47C. A series of sterile tubes are labeled according to the dilution factor. 100 ul of E. coli strain ER2738 overnight culture are added to each of these tubes. Desired serial dilutions are prepared. 10 ul of each dilution are placed in the corresponding culture aliquot, and mixed gently. One aliquot of cells is transferred to an aliquot of top agar, then poured into a small petri dish with no bottom agar. This step is repeated for each aliquot of cells. The top agar is allowed to solidify, then transferred to the 37 degree incubator. Plates are checked for plaques after 8-12 hours.

Nanodropping M13 Phage
Purified phage solutions can be nanodropped to obtain an estimate of the particles/mL. The 269 nm and the 320 nm absorbances are measured, and plugged into the following equations:


SDS-Page of Phage
Phage sample is treated with DNAse and left to incubate for 10 minutes at room temperature. 3x SDS sample buffer with BME is added, then boiled for 10 minutes. Samples are loaded into 14% SDS gel. The gel is run for 50 minutes at 170 V.

Western Blot
A PVDF membrane is soaked in methanol briefly. BioRad Criterion blotter is assembled according to the manual with the gel and the membrane. The blot is run overnight at 20V at 4C with an ice block and a magnetic stirbar. After transfer, the membrane is placed in blocking buffer (1% BSA in 1X TBST) for an hour. The membrane is then washed three times for five minutes in TBST. A primary antibody solution is prepared in .3% BSA in TBST. The membrane is placed in the antibody solution for 1 hour, then washed three times. Probing with the secondary occurs in the same manner. After the wash sequence, the colorimetric substrate solution is added. When bands appear, the reaction is stopped by placing the membrane in water.

AFM Sample Prep
70ul of culture is applied to a freshly cleaved mica surface. The sample is left to sit for 20 minutes, then dipped in Milli-Q water for several seconds to wash. After being dried with nitrogen gas, the sample is imaged.

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