Team:Trieste/protocols
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- | <div id="body"> | + | <div id="body"> |
- | + | <div id="container"> <!-- start container --> | |
- | + | </html>{{Team:Trieste/menu}}<html> | |
- | + | <div id="content"> <!-- start content --> | |
- | + | <h1 id="h1_lf" class="main_tit"><div>Protocols</div></h1> | |
- | + | <h1 id="h1_rt" class="main_tit"><div>More</div></h1> | |
- | + | <div id="box_main"> <!-- start box_main --> | |
- | + | <div class="box_contenuti"> | |
- | + | <div id="preparation" class="notebook_section"> | |
- | + | <h2 class="notebook_title">Preparation of Competent Cells</h2> | |
- | + | Work as sterile as possible at 4°C.</br> | |
- | + | </br> | |
+ | <ol class="fancy"> | ||
+ | <li>Take 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.</li> <li>2. Grow the cells in the shaker at 37°C until they reach an O.D.600nm=0,6.</li> | ||
+ | <li>Transfer them into sterile Falcon (50mL).</li> | ||
+ | <li>Centrifuge it at 4500 rpm for 10 minutes at 4°C. </li> | ||
+ | <li>Resuspend the bacteria pellet on ice in cold CaCl<sub>2</sub>(0,1M) .</li> | ||
+ | <li>Keep this suspension on ice for overnight.</li> | ||
+ | <li>Centrifuge it at 4500 rpm for 10 minutes at 4°C.</li> | ||
+ | <li>Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v).</li> | ||
+ | <li>Dispense in aliquots and freeze cells at -80°C.</li> | ||
+ | </ol> | ||
+ | </div> | ||
- | + | <div id="transformation" class="notebook_section"> | |
+ | <h2 class="notebook_title">Transformation - Heat Shock</h2> | ||
+ | Use DH5-α cells in most cases.<br/> | ||
+ | <br/> | ||
+ | <ol class="fancy"> | ||
+ | <li>Take competent <i>E.coli</i> cells from –80°C freezer and place on ice. Allow cells to thaw.</li> | ||
+ | <li>Mix cells by flicking the tube gently, then remove 100μl per transformation into a sterile pre-chilled (on ice) 1,5 tube.</li> | ||
+ | <li>Add 7μl of DNA per 100μl cells. Quickly flick the tube several times to ensure the even distribution of DNA.</li> | ||
+ | <li>Immediately place tubes on ice for 30 minutes.</li> | ||
+ | <li>Heat shock the cells for 90 seconds in a water bath at exactly 42 °C. Do no shake.</li> | ||
+ | <li>Immediately place tubes on ice for 2 minutes. </li> | ||
+ | <li>Add 1mL of room temperature LB media (with no antibiotic added) and incubate for 1 hour in shaker at 37°C.Can incubate tubes for 30 minutes with appropriate antibiotic added – usually Ampicillin or Kanamycin.</li> | ||
+ | <li>Spread about 100μL of the resulting culture on LB plates - Grow overnight (O/N). | ||
+ | The cells may be pelleted by centrifugation at 500 x g for 5 minutes, then the cells can be resuspended and plated.</li> | ||
+ | <li>Pick colonies about 12-16 hours later.</li> | ||
+ | </ol> | ||
+ | </div> | ||
- | |||
- | |||
- | + | <div id="clean" class="notebook_section"> | |
+ | <h2 class="notebook_title">Clean colony PCR</h2> | ||
+ | <ol class="fancy"> | ||
+ | <li>Pick the bacterial colonies and release them in 50μL distillate/autoclaved water.</li> | ||
+ | <li>Boil the sample at 95°C for 5 minutes.</li> | ||
+ | <li>Prepare 28μL of mix-PCR solution for each sample (6μL Buffer Taq 5x, 1,8μL MgCl2 25mM, 0,6μL dNTPs 5mM, 0,15μL per primers, 0,15μL Taq polymerase, 19,15μL H2O), blend it and then spin it.</li> | ||
+ | <li>Take 2mL of the sample and release into mix-PCR solution and blend it.</li> | ||
+ | <li>Impost the PCR machine for 30μL volume and for 30 cycles, 5 minutes at 93°C, 30 seconds at 95°C, 30 seconds at 53°C, 1 minute at 72°C, indefinitely at 4°C.</li> | ||
+ | <li>Insert the samples and start the PCR machine.</li> | ||
+ | <li>At the end of the PCR the samples are ready for electrophoresis. </li> | ||
+ | </ol> | ||
- | + | </div> | |
- | + | <div id="elisa" class="notebook_section"> | |
+ | <h2 class="notebook_title">E.L.I.S.A.</h2> | ||
+ | <ol class="fancy"> | ||
+ | <li>Coat 96-well ELISA plate with 100μL per well of antibody I anti6HIS used 1μg/mL for selection. Coating is in 100mM sodium hydrogen carbonate, pH 9.6. Leave O/N at 25°C.</li> | ||
+ | <li>Rinse wells 5x with BSA-PBS 0,1%.</li> | ||
+ | <li>Add the bacteria transformed that express the 6HIS tag in different concentration: 10<sup>6</sup>, 10<sup>5</sup>, 10<sup>4</sup> in different wells. Then in different wells too add bacteria non-transformed in different concentration: 10<sup>6</sup>, 10<sup>5</sup>, 10<sup>4</sup>.</li> | ||
+ | <li>Rinse wells 5x with LB media.</li> | ||
+ | <li>Add 200μL of LB media and possibly antibiotics. Incubate O/N at 37°C.</li> | ||
+ | <li>Plate and incubate at 37°C until formation of bacterial colonies.</li> | ||
+ | </ol> | ||
+ | </div> | ||
- | + | <div id="western" class="notebook_section"> | |
+ | <h2 class="notebook_title">Western blotting</h2> | ||
+ | <h3>Preparation</h3> | ||
+ | <ol class="fancy"> | ||
+ | <li>Inoculate bacteria in 20mL of LB media with antibiotics if required O/N.</li> | ||
+ | <li>Transfer 2mL of the inoculum in flask and add 18mL of LB media with antibiotics if required.</li> | ||
+ | <li>Grow the bacteria until the inoculum reach at O.D. 600nm the value 0,4-0,6.</li> | ||
+ | <li>2mL must be recovered to form the sample “non-induced”.</li> | ||
+ | <li>Induce the remaining 17mL of inoculum with 17μL of IPTG.</li> | ||
+ | <li>Wait for 4 hours (or for the time deemed appropriate).</li> | ||
+ | <li>Take 2ml the induced and centrifuge it for 10 minutes at 5000 rcf.</li> | ||
+ | <li>Discard the supernatant and add to the pellet 200μL of Loading Buffer SDS. </li> | ||
+ | <li>Sonicate very strong. </li> | ||
+ | <li>Heat shock at 95°C for 5 minutes.</li> | ||
+ | </ol> | ||
+ | <h3>SDS-PAGE</h3> | ||
+ | <ol class="fancy"> | ||
+ | <li>Assemblate the Western blot scaffold.</li> | ||
+ | <li>Seal the bottom of the Western blot scaffold using agarose-water solution.</li> | ||
+ | <li>Add 10mL the running gel. </li> | ||
+ | <li>Add immediately, before the gel get solid, 1mL of isopropanol to level off the gel surface.</li> | ||
+ | <li>When the running gel is solid, add stacking gel until edge.</li> | ||
+ | <li>Insert the comb and wait the solidification of the gel.</li> | ||
+ | <li>Fill the Western blot scaffold with running buffer.</li> | ||
+ | <li>Remove gently the comb and wash the wells with Loading buffer SDS.</li> | ||
+ | <li>Loading the samples into wells.</li> | ||
+ | <li>Run Western blot at 200V, 30mA for 2 hours.</li> | ||
+ | </ol> | ||
+ | <h3>Transfer</h3> | ||
+ | <ol class="fancy"> | ||
+ | <li>At the end of the SDS-PAGE, disassemble the Western blot scaffold and recover the gel.</li> | ||
+ | <li>Assemblate the sandwich with in the middle the PVDF membrane surrounded two pieces of filter paper.</li> | ||
+ | <li>Put the sandwich into transfer box, add transfer buffer.</li> | ||
+ | <li>Leave transfer proteins at 200V, 50mA O/N.</li> | ||
+ | </ol> | ||
+ | <h3>Blocking</h3> | ||
+ | <ol class="fancy"> | ||
+ | <li>Dip the PVDF membrane into PBS-milk 5%, 1 hours in shaker, to prevent non-specific binding of the antibodies, which leads to high backgrounds.</li> | ||
+ | <li>Discard the PBS-milk 5% solution.</li> | ||
+ | <li>Add Antibody I with PBS-milk 5% solution and incubate for 1 hours in shaker.</li> | ||
+ | <li>Discard antibody with PBS-milk 5% solution.</li> | ||
+ | <li>Wash three times with PBS-tween 0,1% solution shake manually.</li> | ||
+ | <li>Wash three times with PBS-tween 0,1% solution in shaker for 3 minutes each one.</li> | ||
+ | <li>Add antibody II with PBS-milk 5% solution and incubate for 1 hours in shaker.</li> | ||
+ | <li>Repeat step 5 and step 6.</li> | ||
+ | </ol> | ||
+ | <h3>ECL (enhanced chemiluminescence)</h3> | ||
+ | <ol class="fancy"> | ||
+ | <li>Dip the membrane in PBS. </li> | ||
+ | <li>Put 1mL first ECL solution + 1mL second ECL solution on film.</li> | ||
+ | <li>Wet the membrane in solution with the solution on film for some seconds.</li> | ||
+ | <li>Fix the wet membrane in obscure box.</li> | ||
+ | <li>Go to obscure room and lean the photograph plate on the membrane for the time deemed appropriate for to have the right exposure.</li> | ||
+ | <li>Dip the photograph plate into developer solution for 1 minute.</li> | ||
+ | <li>Wash the photograph plate with water.</li> | ||
+ | <li>Dip the photograph plate into fixer solution for 2 minutes.</li> | ||
+ | <li>Dry the photograph plate in stove at 65°C. </li> | ||
+ | </ol> | ||
+ | </div> | ||
- | + | <div id="stripping" class="notebook_section"> | |
+ | <h2 class="notebook_title">Stripping</h2> | ||
+ | <ol class="fancy"> | ||
+ | <li>Wash the photograph plate with PBS-Tween 0,1% 3 times shake manually.</li> | ||
+ | <li>Wash the photograph plate with distillate water.</li> | ||
+ | <li>Add Stripping solution + distillate water in a ratio of 1 to 10 and incubate 30 minutes in shaker at 25°C.</li> | ||
+ | <li>Repeat the point 1.</li> | ||
+ | <li>Blocking with PBS-milk 5% for 30 minutes in shaker at 25°C.</li> | ||
+ | <li>Repeat the point 1.</li> | ||
+ | <li>Start the protocol for ECL (enhanced chemiluminescence).</li> | ||
+ | <li>Should be a photograph plate without any signal.</li> | ||
+ | </ol> | ||
+ | </div> | ||
- | + | <div class="notebook_section"> | |
- | + | <h2 class="notebook_title">Precipitation of Surnatant</h2> | |
- | + | <ol class="fancy"> | |
- | + | <li>Recover 1mL of the sample supernatant.</li> | |
+ | <li>Add 250mL of TCA 50% (trichloroacetic acid).</li> | ||
+ | <li>Incubate in ice for 60 minutes.</li> | ||
+ | <li>Centrifuge it for 10 minutes at 3750 rfc at 4°C.</li> | ||
+ | <li>Discard the supernatant.</li> | ||
+ | <li>Add 1mL of could acetone to eliminate the TCA. </li> | ||
+ | <li>Speed vaac for 10 minutes (sniff the sample to control absence of acetone). </li> | ||
+ | <li>Resuspend the sample in sample buffer SDS 1X (the color change to yellow).</li> | ||
+ | <li>Add 1mL of Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol) to basific the solution (the color should change to blue, if it no happen so add other Tris). </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div class="notebook_section"> | ||
+ | <h2 class="notebook_title">Periplasm Protein Preparation</h2> | ||
+ | <ol class="fancy"> | ||
+ | <li>Inoculate bacteria in 20mL of LB media and possibly antibiotics O/N.</li> | ||
+ | <li>Transfer 2mL of the inoculum in flask and add 18mL of LB media and possibly antibiotics.</li> | ||
+ | <li>Grow the bacteria until the inoculum reach at O.D. <sub>600</sub>=0,4-0,6.</li> | ||
+ | <li>2mL must be recovered to form the sample “non-induced”.</li> | ||
+ | <li>Induce the remaining 17mL of inoculum with 17μL of IPTG mM.</li> | ||
+ | <li>Wait for 4 hours (or for the time deemed appropriate).</li> | ||
+ | <li>Take 2mL of the induced and centrifuge it for 10 minutes at 5000 rpm.</li> | ||
+ | <li>Discard the supernatant and resuspend pellet in 1/40 volume of PPB buffer (200mg/mL sucrose, 1mM EDTA, 30mM Tris-HCl pH 8.0). Keep in ice for 20 minutes.</li> | ||
+ | <li>Spin down cells in centrifuge at 5000 rpm for 15 minutes and collect supernatant into smaller high speed centrifuge tubes.</li> | ||
+ | <li>Resuspend pellet in 1/40 volume of 5mM MgSO<sub>4</sub> buffer. Incubate in ice for 20 minutes.</li> | ||
+ | <li>Transfer samples to small high speed centrifuge tubes.</li> | ||
+ | <li>Spin both peri-prep supernatant and Osmotic shock preparation at 15000 rpm for 15 minutes. </li> | ||
+ | <li>Collect the supernatants, the sample is ready for E.L.I.S.A. </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div class="notebook_section"> | ||
+ | <h2 class="notebook_title">Immunofluorescence assay</h2> | ||
+ | <ol class="fancy"> | ||
+ | <li>Inoculate bacteria in 20mL of LB media with antibiotics if required O/N.</li> | ||
+ | <li>Take 1mL when the O.D.<sub>600</sub> arrived at the bacteria concentration about 5x10<sup>8</sup> units/mL.</li> | ||
+ | <li>Take 1mL and centrifugate 10 minutes at 6000 rpm at 4°C.</li> | ||
+ | <li>Wash the sample with PBS 3 times and centrifuge 10 minutes at 6000 rpm at 4°C.</li> | ||
+ | <li>Block with 1mL of PBS-BSA 1% and centrifuge 10 minutes at 6000 rpm at 4°C.</li> | ||
+ | <li>Discard the supernatant and add the primary antibody anti-HIS6.</li> | ||
+ | <li>Rock the sample for 3 hours at 4°C.</li> | ||
+ | <li>Repeat the point four</li> | ||
+ | <li>Discard the supernatant and add the secondary antibody anti-mouse TRICT.</li> | ||
+ | <li>Rock the sample for 1 hour at 4°C.</li> | ||
+ | <li>Repeat the point seven.</li> | ||
+ | <li>Discard the supernatant and add DAPI.</li> | ||
+ | <li>Repeat the point 7.</li> | ||
+ | <li>Analyze the sample with epifluorescence microscope.</li> | ||
+ | </ol> | ||
+ | </div> | ||
</div> | </div> | ||
- | + | </div> <!-- end box_main --> | |
- | + | <div id="box_right"> <!-- start box_right --> | |
- | + | <ul id="sub_menu"> | |
- | + | <li><a href="#preparation">Preparation of Competent Cells</a></li> | |
- | + | <li><a href="#transformation">Transformation - Heat Shock</a></li> | |
- | + | <li><a href="#clean">Clean Colony PCR</a></li> | |
- | + | <li><a href="#elisa">E.L.I.S.A.</a></li> | |
- | + | <li><a href="#western">Western blotting</a></li> | |
- | + | <li><a href="#stripping">Stripping</a></li> | |
- | + | <li><a href="#stripping">Precipitation of Surnatant</a></li> | |
- | + | <li><a href="#stripping">Periplasm Protein Preparation</a></li> | |
- | + | </ul> | |
- | + | </ul> | |
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Latest revision as of 18:30, 26 October 2012
Protocols
More
Preparation of Competent Cells
Work as sterile as possible at 4°C.- Take 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.
- 2. Grow the cells in the shaker at 37°C until they reach an O.D.600nm=0,6.
- Transfer them into sterile Falcon (50mL).
- Centrifuge it at 4500 rpm for 10 minutes at 4°C.
- Resuspend the bacteria pellet on ice in cold CaCl2(0,1M) .
- Keep this suspension on ice for overnight.
- Centrifuge it at 4500 rpm for 10 minutes at 4°C.
- Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v).
- Dispense in aliquots and freeze cells at -80°C.
Transformation - Heat Shock
Use DH5-α cells in most cases.- Take competent E.coli cells from –80°C freezer and place on ice. Allow cells to thaw.
- Mix cells by flicking the tube gently, then remove 100μl per transformation into a sterile pre-chilled (on ice) 1,5 tube.
- Add 7μl of DNA per 100μl cells. Quickly flick the tube several times to ensure the even distribution of DNA.
- Immediately place tubes on ice for 30 minutes.
- Heat shock the cells for 90 seconds in a water bath at exactly 42 °C. Do no shake.
- Immediately place tubes on ice for 2 minutes.
- Add 1mL of room temperature LB media (with no antibiotic added) and incubate for 1 hour in shaker at 37°C.Can incubate tubes for 30 minutes with appropriate antibiotic added – usually Ampicillin or Kanamycin.
- Spread about 100μL of the resulting culture on LB plates - Grow overnight (O/N). The cells may be pelleted by centrifugation at 500 x g for 5 minutes, then the cells can be resuspended and plated.
- Pick colonies about 12-16 hours later.
Clean colony PCR
- Pick the bacterial colonies and release them in 50μL distillate/autoclaved water.
- Boil the sample at 95°C for 5 minutes.
- Prepare 28μL of mix-PCR solution for each sample (6μL Buffer Taq 5x, 1,8μL MgCl2 25mM, 0,6μL dNTPs 5mM, 0,15μL per primers, 0,15μL Taq polymerase, 19,15μL H2O), blend it and then spin it.
- Take 2mL of the sample and release into mix-PCR solution and blend it.
- Impost the PCR machine for 30μL volume and for 30 cycles, 5 minutes at 93°C, 30 seconds at 95°C, 30 seconds at 53°C, 1 minute at 72°C, indefinitely at 4°C.
- Insert the samples and start the PCR machine.
- At the end of the PCR the samples are ready for electrophoresis.
E.L.I.S.A.
- Coat 96-well ELISA plate with 100μL per well of antibody I anti6HIS used 1μg/mL for selection. Coating is in 100mM sodium hydrogen carbonate, pH 9.6. Leave O/N at 25°C.
- Rinse wells 5x with BSA-PBS 0,1%.
- Add the bacteria transformed that express the 6HIS tag in different concentration: 106, 105, 104 in different wells. Then in different wells too add bacteria non-transformed in different concentration: 106, 105, 104.
- Rinse wells 5x with LB media.
- Add 200μL of LB media and possibly antibiotics. Incubate O/N at 37°C.
- Plate and incubate at 37°C until formation of bacterial colonies.
Western blotting
Preparation
- Inoculate bacteria in 20mL of LB media with antibiotics if required O/N.
- Transfer 2mL of the inoculum in flask and add 18mL of LB media with antibiotics if required.
- Grow the bacteria until the inoculum reach at O.D. 600nm the value 0,4-0,6.
- 2mL must be recovered to form the sample “non-induced”.
- Induce the remaining 17mL of inoculum with 17μL of IPTG.
- Wait for 4 hours (or for the time deemed appropriate).
- Take 2ml the induced and centrifuge it for 10 minutes at 5000 rcf.
- Discard the supernatant and add to the pellet 200μL of Loading Buffer SDS.
- Sonicate very strong.
- Heat shock at 95°C for 5 minutes.
SDS-PAGE
- Assemblate the Western blot scaffold.
- Seal the bottom of the Western blot scaffold using agarose-water solution.
- Add 10mL the running gel.
- Add immediately, before the gel get solid, 1mL of isopropanol to level off the gel surface.
- When the running gel is solid, add stacking gel until edge.
- Insert the comb and wait the solidification of the gel.
- Fill the Western blot scaffold with running buffer.
- Remove gently the comb and wash the wells with Loading buffer SDS.
- Loading the samples into wells.
- Run Western blot at 200V, 30mA for 2 hours.
Transfer
- At the end of the SDS-PAGE, disassemble the Western blot scaffold and recover the gel.
- Assemblate the sandwich with in the middle the PVDF membrane surrounded two pieces of filter paper.
- Put the sandwich into transfer box, add transfer buffer.
- Leave transfer proteins at 200V, 50mA O/N.
Blocking
- Dip the PVDF membrane into PBS-milk 5%, 1 hours in shaker, to prevent non-specific binding of the antibodies, which leads to high backgrounds.
- Discard the PBS-milk 5% solution.
- Add Antibody I with PBS-milk 5% solution and incubate for 1 hours in shaker.
- Discard antibody with PBS-milk 5% solution.
- Wash three times with PBS-tween 0,1% solution shake manually.
- Wash three times with PBS-tween 0,1% solution in shaker for 3 minutes each one.
- Add antibody II with PBS-milk 5% solution and incubate for 1 hours in shaker.
- Repeat step 5 and step 6.
ECL (enhanced chemiluminescence)
- Dip the membrane in PBS.
- Put 1mL first ECL solution + 1mL second ECL solution on film.
- Wet the membrane in solution with the solution on film for some seconds.
- Fix the wet membrane in obscure box.
- Go to obscure room and lean the photograph plate on the membrane for the time deemed appropriate for to have the right exposure.
- Dip the photograph plate into developer solution for 1 minute.
- Wash the photograph plate with water.
- Dip the photograph plate into fixer solution for 2 minutes.
- Dry the photograph plate in stove at 65°C.
Stripping
- Wash the photograph plate with PBS-Tween 0,1% 3 times shake manually.
- Wash the photograph plate with distillate water.
- Add Stripping solution + distillate water in a ratio of 1 to 10 and incubate 30 minutes in shaker at 25°C.
- Repeat the point 1.
- Blocking with PBS-milk 5% for 30 minutes in shaker at 25°C.
- Repeat the point 1.
- Start the protocol for ECL (enhanced chemiluminescence).
- Should be a photograph plate without any signal.
Precipitation of Surnatant
- Recover 1mL of the sample supernatant.
- Add 250mL of TCA 50% (trichloroacetic acid).
- Incubate in ice for 60 minutes.
- Centrifuge it for 10 minutes at 3750 rfc at 4°C.
- Discard the supernatant.
- Add 1mL of could acetone to eliminate the TCA.
- Speed vaac for 10 minutes (sniff the sample to control absence of acetone).
- Resuspend the sample in sample buffer SDS 1X (the color change to yellow).
- Add 1mL of Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol) to basific the solution (the color should change to blue, if it no happen so add other Tris).
Periplasm Protein Preparation
- Inoculate bacteria in 20mL of LB media and possibly antibiotics O/N.
- Transfer 2mL of the inoculum in flask and add 18mL of LB media and possibly antibiotics.
- Grow the bacteria until the inoculum reach at O.D. 600=0,4-0,6.
- 2mL must be recovered to form the sample “non-induced”.
- Induce the remaining 17mL of inoculum with 17μL of IPTG mM.
- Wait for 4 hours (or for the time deemed appropriate).
- Take 2mL of the induced and centrifuge it for 10 minutes at 5000 rpm.
- Discard the supernatant and resuspend pellet in 1/40 volume of PPB buffer (200mg/mL sucrose, 1mM EDTA, 30mM Tris-HCl pH 8.0). Keep in ice for 20 minutes.
- Spin down cells in centrifuge at 5000 rpm for 15 minutes and collect supernatant into smaller high speed centrifuge tubes.
- Resuspend pellet in 1/40 volume of 5mM MgSO4 buffer. Incubate in ice for 20 minutes.
- Transfer samples to small high speed centrifuge tubes.
- Spin both peri-prep supernatant and Osmotic shock preparation at 15000 rpm for 15 minutes.
- Collect the supernatants, the sample is ready for E.L.I.S.A.
Immunofluorescence assay
- Inoculate bacteria in 20mL of LB media with antibiotics if required O/N.
- Take 1mL when the O.D.600 arrived at the bacteria concentration about 5x108 units/mL.
- Take 1mL and centrifugate 10 minutes at 6000 rpm at 4°C.
- Wash the sample with PBS 3 times and centrifuge 10 minutes at 6000 rpm at 4°C.
- Block with 1mL of PBS-BSA 1% and centrifuge 10 minutes at 6000 rpm at 4°C.
- Discard the supernatant and add the primary antibody anti-HIS6.
- Rock the sample for 3 hours at 4°C.
- Repeat the point four
- Discard the supernatant and add the secondary antibody anti-mouse TRICT.
- Rock the sample for 1 hour at 4°C.
- Repeat the point seven.
- Discard the supernatant and add DAPI.
- Repeat the point 7.
- Analyze the sample with epifluorescence microscope.