|
|
| (43 intermediate revisions not shown) |
| Line 3: |
Line 3: |
| | {{:CSS/gifu/protocols}} | | {{:CSS/gifu/protocols}} |
| | {{:CSS/gifu/tab}} | | {{:CSS/gifu/tab}} |
| - |
| |
| - |
| |
| | <html> | | <html> |
| | <head> | | <head> |
| Line 10: |
Line 8: |
| | <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.9.1/jquery.min.js" type="text/javascript"> | | <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.9.1/jquery.min.js" type="text/javascript"> |
| | </script> | | </script> |
| - |
| |
| | <script type="text/javascript"> | | <script type="text/javascript"> |
| | <!-- | | <!-- |
| | $(document).ready(function(){ | | $(document).ready(function(){ |
| | $("tr:even").addClass("odd"); | | $("tr:even").addClass("odd"); |
| - |
| |
| - | });
| |
| - | //-->
| |
| - | </script>
| |
| | | | |
| | </head> | | </head> |
| | <body> | | <body> |
| - |
| |
| | <div id="tab"> | | <div id="tab"> |
| - | <a class="tab-white" href="https://2014.igem.org/Team:Gifu/Protocol">Protocols</a> | + | <a class="tab-white" href="https://2014.igem.org/Team:Gifu/Protocol">Protocols 1</a> |
| | + | <a class="tab-gray" href="https://2014.igem.org/Gifu/protocols2">Protocols 2</a> |
| | <a class="tab-gray" href="https://2014.igem.org/Team:Gifu/Notebook" >Calender</a></div> | | <a class="tab-gray" href="https://2014.igem.org/Team:Gifu/Notebook" >Calender</a></div> |
| - |
| |
| | <div id="main"> | | <div id="main"> |
| | <h1 id="theme"><a name="top"></a>Protocols</h1> | | <h1 id="theme"><a name="top"></a>Protocols</h1> |
| | <ul class="list"> | | <ul class="list"> |
| | + | |
| | + | <li><a href="#RD">Media</a></li> |
| | <li><a href="#RD">Restriction Digests</a></li> | | <li><a href="#RD">Restriction Digests</a></li> |
| | <li><a href="#LI">Ligation</a></li> | | <li><a href="#LI">Ligation</a></li> |
| Line 37: |
Line 31: |
| | <li><a href="#EL">Electrophoresis</a></li> | | <li><a href="#EL">Electrophoresis</a></li> |
| | <li><a href="#MI">Miniprep</a></li> | | <li><a href="#MI">Miniprep</a></li> |
| - | <li><a href="#GE">Gel Extraction with FastGene™ Gel/PCR Extraction Kit</a></li> | + | <li><a href="#SDS">SDS-PAGE</a></li> |
| - | <li><a href="#PCRP">PCR Purification with FastGene™ Gel/PCR Extraction Kit</a></li>
| + | |
| - | <li><a href="#PP">Prorein Purification by use of Ni-NTA</a></li>
| + | |
| - | <li><a href="#HS">HisLink Spin Protein Purification</a></li>
| + | |
| - | <li><a href="#RE">RNA Extraction using Pure Yield™RNA Midiprep System (Promega)</a></li>
| + | |
| - | <li><a href="#WB">Western blotting</a></li>
| + | |
| | </ul> | | </ul> |
| | + | |
| | + | |
| | <div id="contents"> | | <div id="contents"> |
| | <h2><a name="RD"></a>Restriction Digests</h2> | | <h2><a name="RD"></a>Restriction Digests</h2> |
| | + | <h4>Materials</h4> |
| | + | <ul><li>Ice and bucket/container</li> |
| | + | <li>DNA to be digested</li> |
| | + | <li>Buffer M (10x)</li> |
| | + | <li>Restriction Enzymes: EcoRI, SpeI, XbaI, PstI</li> |
| | + | <li>Incubator</li> |
| | + | </ul> |
| | + | <h4>Procedure(an example)</h4> |
| | + | <ol><li>Add 47ul of DNA to be digested into a 1.5ml microcentrifuge tube.</li> |
| | + | <li>Add 5.0ul of10x buffer M.</li> |
| | + | <li>Add 1.0ul of EcoRI.</li> |
| | + | <li>Add 1.0ul of SpeI.</li> |
| | + | <li>There should be a total volume of 50ul. Mix well and spin down briefly.</li> |
| | + | <li>Incubate the restriction digest at 38C for 30min. We incubate in an incubator.</li> |
| | + | <li>Run a portion of the digest on a gel (6ul), to check that part length is accurate.</li> |
| | + | </ol> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| | + | </noscript> |
| | + | |
| | <h2><a name="LI"></a>Ligation</h2> | | <h2><a name="LI"></a>Ligation</h2> |
| | + | <ol><li>Add digested fragment A.</li> |
| | + | <li>Add digested fragment B.</li> |
| | + | <li>Add ligation mixture.</li> |
| | + | <li>Ligate 16C/30 min.</li> |
| | + | </ol> |
| | + | <p><em>Note:</em> Make the amount of fragment A and B equimolar. And make sure that the volume of ligation mixture is equivalent to the total amount of the volume of fragment A and B. we used a constant temperature water bath.</p> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| | + | </noscript> |
| | + | |
| | <h2><a name="TF"></a>Transformation</h2> | | <h2><a name="TF"></a>Transformation</h2> |
| | + | <ol><li>Deal 25ul of competent cells to each 1.5ml tubes.</li> |
| | + | <li>Add 1ul of each solution made by ligation to each tube.</li> |
| | + | <li>Close tubes and incubate the cells for 30 min on ice.</li> |
| | + | <li>Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 seconds.</li> |
| | + | <li>Incubate the cells on ice for 2 minutes.</li> |
| | + | <li>Add 225ul of SOC media to each tube.</li> |
| | + | <li>Incubate cells at 37C for 30 minutes.</li> |
| | + | <li>Inoculate with each solution of cells to each plate with appropriate antibiotics.</li> |
| | + | <li>Culture the cells.</li></ol> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| - | <h2><a name="DE"></a>Densitometry(Nanodrop)</h2> | + | </noscript> |
| - | <p align="right"><a href="#top">back to page top</a></p>
| + | |
| - | <h2><a name="CP"></a>Colony PCR</h2>
| + | <h2><a name="DE"></a>Densitometry(Nanodrop,In case of nucleic acid)</h2> |
| - | <p align="right"><a href="#top">back to page top</a></p>
| + | |
| - | <h2><a name="EL"></a>Electrophoresis</h2>
| + | |
| - | <p align="right"><a href="#top">back to page top</a></p>
| + | |
| - | <h2><a name="MI"></a>Miniprep</h2>
| + | |
| - | <p align="right"><a href="#top">back to page top</a></p>
| + | |
| - | <h2><a name="GE"></a>Gel Extraction with FastGene™ Gel/PCR Extraction Kit</h2>
| + | |
| - | <h4>Separation of the gel</h4>
| + | |
| | <ol> | | <ol> |
| | <li> | | <li> |
| - | Cut down a DNA fragment from an agarose gel. Remove surplus agarose to make the gel fragment as small as ossible.</br>
| + | Activate the device. |
| - | Note: Recommended concentration of agarose is under 2.5%.
| + | |
| | </li> | | </li> |
| | <li> | | <li> |
| - | Transfer the gel fragment(max 300 mg) to a centrifugeal tube.
| + | Choose “Nucleic Acid” at main menu. |
| | </li> | | </li> |
| | <li> | | <li> |
| - | Add 500 µL of buffer for combination GP1 to a sample and then stir it with a vortex mixer.
| + | Wash a space of a sample with distilled water and then wipe gently the top and bottom of the arm. |
| | </li> | | </li> |
| | <li> | | <li> |
| - | Incubate a sample at 55 C° for 10~15 minutes (until the gel fragment completely dissolves). While incubating invert the tube every 2~3 minutes.
| + | Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA. |
| | </li> | | </li> |
| - | </ol>
| |
| - | <h4>DNA bonding</h4>
| |
| - | <ol>
| |
| | <li> | | <li> |
| - | Insert FastGene™GP column into a collection tube.
| + | Pour distilled water to test a blank and after that wipe the top and bottom of the arm.</li> |
| - | </li> | + | |
| | <li> | | <li> |
| - | Dispense (max 800 µL of) sample solution of the previous step into FastGene™GP columns and then centrifuge them(13,000 rpm 30 seconds).
| + | Pour the sample to measure the concentration. |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Throw filtrate away and then return the column to the collection tube.</br>
| + | |
| - | Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.
| + | |
| | </li> | | </li> |
| | </ol> | | </ol> |
| - | <h4>Washing</h4> | + | <noscript> |
| | + | <p align="right"><a href="#top">back to page top</a></p> |
| | + | </noscript> |
| | + | |
| | + | <h2><a name="CP"></a>Colony PCR</h2> |
| | <ol> | | <ol> |
| - | <li> | + | <li>Collect a colony from a plate and then suspend it in 100 µL of LB media (liquid). </li> |
| - | Add 600 µL of washing buffer GP2 to a FastGene™GP column and then centrifuge it(13,000 rpm 30 seconds).
| + | <li>Mix the following reagents.</br> |
| - | </li> | + | <table class="table" border="2"> |
| - | <li> | + | <tr><td>sterile water</td><td>27.5 µL</td></tr> |
| - | Throw filtrate away and then return the column to the collection tube.</br>
| + | <tr><td>10×PCR buffer</td><td>5 µL</td></tr> |
| - | Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat the step of Washing to completely remove boric acid.
| + | <tr><td>2 mM dNTP</td><td>5 µL</td></tr> |
| - | </li> | + | <tr><td>5 pmol/µL Fw primer</td><td>2.5 µL</td></tr> |
| - | </ol> | + | <tr><td>5 pmol/µL Rv primer</td><td>2.5 µL</td></tr> |
| - | <h4>Desiccation</h4> | + | <tr><td>0.5U/µL Taq polymerase</td><td>2.5 µL</td></tr> |
| - | <ol> | + | <tr><td>total</td><td>45 µL</td></tr> |
| - | <li> | + | </table></li> |
| - | Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.
| + | <li>Add 5 µL of the suspension to the mixture of reagents.</li> |
| - | </li> | + | <li>Do PCR amplification.</li> |
| - | </ol> | + | <li>Measure the length of the DNA.</li> |
| - | <h4>DNA Elution</h4> | + | |
| - | <ol> | + | |
| - | <li> | + | |
| - | Insert a FastGene™GP column into a new centrifugeal tube.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Add 20~50 µL of elution buffer GP3 to the center of column matrix. | + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Until elution buffer is absorbed by the column matrix, keep the same state for 2 minutes.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.</br>
| + | |
| - | Caution: The yield of a large DNA fragment(more than 5 kbp) can be increased by the use of advance heated(70 C°) buffer.
| + | |
| - | </li> | + | |
| | </ol> | | </ol> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| - | <h2><a name="#PCRP"></a>PCR Purification with FastGene™ Gel/PCR Extraction Kit</h2> | + | </noscript> |
| - | <h4>Sample preparation</h4> | + | |
| | + | <h2><a name="EL"></a>Electrophoresis (preparing 200 mL of agarose solution)</h2> |
| | + | <p> |
| | <ol> | | <ol> |
| - | <li> | + | <li>Meter 4 g of agarose.</li> |
| - | Add buffer for combination GP1 to a sample in the ratio of one to five(e.g. 200 µL of buffer for combination GP1 per 40 µL of PCR reaction liquid).</br>
| + | <li>Add 200 mL of 1× buffer into it.</li> |
| - | Caution: If the volume of the sample is under 40 µL, add buffer for combination GP1 or water for PCR to adjust the volume of the sample to 40 µL.
| + | <li>Wrap the neck of flask and then make a hole on the wrap.</li> |
| - | </li> | + | <li>Dissolve the agarose with a microwave oven.</li> |
| - | <li> | + | <li>Cool it to a suitable temperature.</li> |
| - | Stir it with a vortex mixer.
| + | <li>Pour the agarose solution into a mold with a comb.</li> |
| - | </li> | + | <li>Remove bubbles.</li> |
| - | <li> | + | <li>After the gel going solid, dislodge the comb carefully.</li> |
| - | If the gross of volume of buffer for combination GP1 and water for PCR exceed the capacity of a PCR tube, transfer water for PCR to a centrifugeal tube.
| + | <li>Transfer the mold into a phoresis tank.</li> |
| - | </li> | + | <li>Immerse the mold in 1× buffer.</li> |
| - | </ol> | + | <li>Mix 5 µL of DNA solution and 1 µL of 3× dye.</li> |
| - | <h4>DNA bonding</h4>
| + | <li>Pour the mixture into the well.</li> |
| - | <ol>
| + | <li>Electrophorese at 100V.</li> |
| - | <li> | + | <li>Stop the electrophoresis when the band of dye go up to 3/4 of the gel.</li> |
| - | Insert FastGene™GP column into a collection tube.
| + | <li>Pick out the gel and then stain it with ethidium bromide (0.5 µg/mL) for 20 minutes.</li> |
| - | </li> | + | <li>Observe DNA bands with UV transilluminator.</li> |
| - | <li> | + | |
| - | Dispense the sample solution of the previous step into FastGene™GP columns and then centrifuge them(13,000 rpm 30 seconds).
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Throw filtrate away and then return the column to the collection tube.
| + | |
| - | </li> | + | |
| - | </ol> | + | |
| - | <h4>Washing</h4> | + | |
| - | <ol>
| + | |
| - | <li> | + | |
| - | Add 600 µL of washing buffer GP2 to a FastGene™GP column and then centrifuge it(13,000 rpm 30 seconds).
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Throw filtrate away and then return the column to the collection tube.
| + | |
| - | </li> | + | |
| - | </ol>
| + | |
| - | <h4>Desiccation</h4>
| + | |
| - | <ol>
| + | |
| - | <li> | + | |
| - | Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.
| + | |
| - | </li> | + | |
| - | </ol> | + | |
| - | <h4>DNA Elution</h4>
| + | |
| - | <ol>
| + | |
| - | <li>
| + | |
| - | Insert a FastGene™GP column into a new centrifugeal tube.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Add 20~50 µL of elution buffer GP3 to the center of column matrix.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Until elution buffer is absorbed by the column matrix, keep the same state for 2 minutes.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.</br>
| + | |
| - | Caution: The yield of a large DNA fragment(more than 5 kbp) can be increased by the use of advance heated(70 C°) buffer.
| + | |
| - | </li> | + | |
| | </ol> | | </ol> |
| | + | </p> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| - | <h2><a name="PP"></a>Prorein Purification with Ni-NTA</h2> | + | </noscript> |
| - | <br><br><br> | + | |
| - | <!--
| + | <h2><a name="MI"></a>Miniprep</h2> |
| - | できてない！
| + | <noscript> |
| - | -->
| + | |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| - | <h2><a name="HS"></a>HisLink Spin Protein Purification</h2>
| + | </noscript> |
| - | <h4>Materials to Be Supplied by the User</h4>
| + | |
| - | <ul>
| + | |
| - | <li>Nuclease-Free Water</li>
| + | |
| - | <li>rotor</li>
| + | |
| - | <li>wide-bore pipette tips</li>
| + | |
| - | <li>NaCl or 5M NaCl solution for use with HQ-tagged proteins</li>
| + | |
| - | <li>tabletop centrifuge</li>
| + | |
| - | <li>1.5ml microcentrifuge tubes</li>
| + | |
| - | </ul>
| + | |
| - | <h4>Centrifugation Protocol</h4>
| + | |
| - | <ol>
| + | |
| - | <li>
| + | |
| - | Pipet 700μl of bacterial culture into a 1.5ml microcentrifuge tube. Add 70μl
| + | |
| - | of the FastBreak™ Reagent/DNase I solution prepared in Section 3.D,
| + | |
| - | Table 1, to the culture.</br>
| + | |
| - | Note: For HQ-tagged proteins, please see Section 3.C.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Resuspend the resin and allow it to settle. Once the resin has settled, use a
| + | |
| - | wide-bore pipette tip to transfer 75μl of the HisLink™ Resin from the
| + | |
| - | settled resin bed to the 1.5ml microcentrifuge tube. To successfully transfer
| + | |
| - | resin, place the wide-bore pipette tip deep into the resin and pipet slowly
| + | |
| - | to assure that a consistent amount of resin is drawn into the pipette. Allow
| + | |
| - | the resin to resettle between each pipetting.</br>
| + | |
| - | Note: We recommend optimizing the amount of HisLink™ Resin used for
| + | |
| - | low- (<1mg/ml) or high- (>1mg/sample) expressing proteins. For lowexpressing
| + | |
| - | proteins, less resin should be used; similarly, for highexpressing
| + | |
| - | proteins, more resin per sample can be used.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Incubate the sample and resin for 30 minutes, mixing frequently on a
| + | |
| - | rotating platform or shaker to optimize binding.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Place a Spin Column onto a Collection Tube (or a new
| + | |
| - | 1.5ml microcentrifuge tube). Use a wide-bore pipette tip to transfer the
| + | |
| - | lysate and resin from the original 1.5ml microcentrifuge tube in Step 3 to
| + | |
| - | the spin column. If resin remains in the 1.5ml microcentrifuge tube, add
| + | |
| - | HisLink™ Binding/Wash Buffer to the tube, then transfer the buffer and
| + | |
| - | remaining resin to the spin column.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Centrifuge the spin column with the collection tube for 5 seconds or until
| + | |
| - | the liquid clears the spin column.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | To save the flowthrough, remove the spin column from the collection
| + | |
| - | tube and transfer the flowthrough from the collection tube to a new
| + | |
| - | 1.5ml microcentrifuge tube. Otherwise, discard the flowthrough.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Place the spin column back onto the collection tube. Add 500μl of
| + | |
| - | HisLink™ Binding/Wash Buffer to the spin column, then cap the spin
| + | |
| - | column. Centrifuge for 5 seconds or until the Binding/Wash Buffer clears
| + | |
| - | the spin column. Discard the flowthrough. Repeat for a total of two washes.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Take the spin column off the collection tube and wipe the base of the spin
| + | |
| - | column with a clean absorbent paper towel to remove any excess
| + | |
| - | HisLink™ Binding/Wash Buffer.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Place the spin column onto a new 1.5ml microcentrifuge tube. Add 200μl of
| + | |
| - | HisLink™ Elution Buffer. Cap the spin column and tap or flick it several
| + | |
| - | times to resuspend the resin. Wait 3 minutes.</br>
| + | |
| - | Note: HQ-tagged proteins may elute with a lower concentration of
| + | |
| - | imidazole (50–100mM) compared to polyhistidine-tagged proteins.
| + | |
| - | Section 4.A has details on the use of imidazole for elution.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Centrifuge the spin column and microcentrifuge tube at 14,000rpm for
| + | |
| - | 1 minute to collect the eluted protein.
| + | |
| - | </li>
| + | |
| | | | |
| - | </ol>
| + | <h2><a name="SDS"></a>SDS-PAGE</h2> |
| - | <p align="right"><a href="#top">back to page top</a></p>
| + | <h4>Protein extraction and Sample preparation</h4> |
| - | <h2><a name="RE"></a>RNA Extraction with Pure Yield™RNA Midiprep System (Promega)</h2> | + | |
| - | <h4>Sample Preparation(E.coli)</h4> | + | |
| - | <h7>
| + | |
| - | <p>Materials to Be Supplied by the User</p>
| + | |
| - | <ul>
| + | |
| - | <li>TE buffer</li>
| + | |
| - | <li>lysozyme(0.4 mg Lysozyme/ mL TE buffer)</li>
| + | |
| - | <li>Lysis Solution(20 µL β-Mercaptoethanol/ mL RNA Lysis Buffer)</li>
| + | |
| - | <li>RNA Wash Solution(206 mL RNA Wash Solution + 350 mL 95% ethanol)</li>
| + | |
| - | </ul>
| + | |
| - | </h7>
| + | |
| | <ol> | | <ol> |
| - | <li> | + | <li>Add 50 µL of culture fluid that was cultured overnight to a liquid culture medium, and then cultivate it for a few hours.</li> |
| - | Inoculate 10ml of growth medium with a single colony of your bacterial
| + | <li>When the bacteria are not too many, add a proper quantity of IPTG to the liquid culture medium.</li> |
| - | sample. Incubate overnight at the appropriate temperature with shaking.
| + | <li>Cultivate it for a few hours and then store it at low temperature.</li> |
| - | Do not use the overnight culture directly for RNA isolation; instead use it
| + | <li>Pour 1 mL of the liquid culture into a 1.5ml tube. Centrifuge it(13,000 rpm 4°C 15 minutes) and then discard supernatant fluid. Do twice this step. </li> |
| - | to inoculate a fresh culture as described in Step 2.
| + | <li>Add 300 µL of PBS to the deposition and then stir it.</li> |
| - | </li> | + | <li>Sonicate the cell suspension with 4 short bursts of 15 sec followed by intervals of 60 sec for cooling.</li> |
| - | <li> | + | <li>Centrifuge(13,000rpm, 4°C, 20minutes)and then collect supernatant into another tube.</li> |
| - | Dilute the overnight culture 1:50 and grow to an optical density (O.D.600) of
| + | <li>Add 100 ul of PBS to the deposition. Shake it with a vortex and then collect it.</li> |
| - | 0.6–1.0 by spectrophotometry. This should only take a few hours. If growth
| + | <li>Pour 8 µL of the supernatant(=cell extract) into a tube, and 8 µL of the precipitation suspension into another tube.</li> |
| - | is too slow, reduce the dilution factor by adding a larger volume of
| + | <li>Add 2 µL of 5×loading dye to the tube and then denature it at 90°C with a block incubator.</li> |
| - | inoculum to fresh medium.</br>
| + | <li>Centrifuge and adjust the sample.</li> |
| - | Note: The doubling time of E. coli is approximately 20 minutes in rich
| + | <li>Apply 10µL of the sample to SDS gel. </li> |
| - | medium. The growth rate depends on the growth medium, temperature,
| + | |
| - | strain and degree of oxygenation. For most purposes, the culture should be
| + | |
| - | harvested in mid-log phase. A stationary phase E. coli culture generally
| + | |
| - | contains 1–2 × 109 cells/ml, depending on the culture medium and
| + | |
| - | aeration.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Transfer a volume of culture containing a maximum of 1 × 1010 cells to a
| + | |
| - | centrifuge tube. Centrifuge at 5,500 × g for 5 minutes at 4°C. Carefully
| + | |
| - | remove the supernatant, leaving the cell pellet as dry as possible.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Resuspend the cell pellet in 1ml of freshly prepared TE buffer containing
| + | |
| - | lysozyme. Do not exceed 1ml per prep. Tap gently to mix.</br>
| + | |
| - | Note: For Gram-positive bacteria use 3mg/ml lysozyme; for Gram-negative
| + | |
| - | bacteria, use 0.4mg/ml lysozyme.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Incubate the resuspended cells at room temperature. For Gram-positive
| + | |
| - | bacteria, incubate the cells for 5–10 minutes; for Gram-negative bacteria,
| + | |
| - | incubate the cells for 3–5 minutes.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Add 1ml of Lysis Solution containing BME. Vortex vigorously until lysis is | + | |
| - | complete and no clumps are present. Incubate on ice for 10 minutes to
| + | |
| - | complete the lysis.</br>
| + | |
| - | Optional: Homogenize the lysate at high speed, using a rotor-stator
| + | |
| - | homogenizer (such as a BioSpec Tissue-Tearor™), until no visible clumps
| + | |
| - | remain.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Proceed to Lysate Clearing.
| + | |
| - | </li> | + | |
| | </ol> | | </ol> |
| - | <h4>Lysate Clearing </h4> | + | |
| | + | <h4>Making a SDS-PAGE gel and Electrophpresis</h4> |
| | <ol> | | <ol> |
| - | <li> | + | <li>Mix and shake quickly reagents to adjust the concentration of separation gel.</li> |
| - | Transfer 2ml of lysate to a 15ml, capped, disposable centrifuge tube. Lysate
| + | <li>Construct a plate for electrophoresis.</li> |
| - | volumes larger than 2ml may be processed with multiple columns or the
| + | <li>Pour separation gel into a gap of the plate (until about 2 cm below a comb).</br> |
| - | remainder can be stored at –70°C for later use. If the volume of lysate is not a
| + | Note: Wipe a plate for electrophoresis with 70% ethanol.</br> |
| - | multiple of 2ml, add an appropriate volume of Lysis Solution to achieve 2ml
| + | Hold a plate for electrophoresis not to spill the gel.</br> |
| - | per prep.
| + | <li>Pour a proper quantity of Milli Q water into a gap of the plate and then incubate for an hour.</li> |
| - | </li> | + | <li>Mix and shake quickly reagents for stacking gel (except APS and TEMED).</li> |
| - | <li> | + | <li>Slant the gel plate and absorb multistoried Milli Q water.</li> |
| - | Add 4ml of RNA Dilution Buffer (blue) to 2ml of lysate. Immediately seal the
| + | <li>Add APS and TEMED to mixed reagents for stacking gel (step5).</li> |
| - | tube, and mix thoroughly by inverting the tube 3–4 times and then vortexing.
| + | <li>Fill the gap of the plate with stacking gel and then insert the comb into the gap of the plate.</br> |
| - | Process all samples before continuing.
| + | Note: Be careful not to mix bubbles in gel.</li> |
| - | </li> | + | <li>Take out the plate and gel together after stacking gel coagulates.</li> |
| - | <li> | + | <li>Put the plate and gel into a migration tank with the plate toward outside.</li> |
| - | Thoroughly mix the white Clearing Agent by shaking or vortexing the bottle
| + | <li>Pour 300 µL of electrophoresis buffer into a phoresis tank. Immerse the gel completely.</li> |
| - | until all of the material is in suspension. Mix until no material adheres to the
| + | <li>Apply 10 µL of the sample and 5 µL of a marker.</li> |
| - | bottom of the bottle when inverted. This may take several minutes.</br>
| + | <li>Electrophorese at 40 mA in the stacking gel and then at 60mA in the separation gel.</li> |
| - | Note: The Clearing Agent settles over time and should be remixed between | + | <li>Electrophorese until a pigment comes at an appropriate position.</li> |
| - | samples. When the Clearing Agent is mixed properly, it will not clog pipet tips.
| + | <li>Stop electrophoresis and then carefully take the gel.</br> |
| - | </li> | + | Note: Use tweezers.</li> |
| - | <li> | + | <li>Discard the buffer for electrophoresis and then dye the separation gel with CBB.</li> |
| - | Add 1ml of Clearing Agent to the diluted lysate. Immediately seal the tube,
| + | <li>Wash the gel plate and the electrophoretic tank with neutral detergent and then rinse it steadily.</li> |
| - | and mix by inverting 2–3 times and vortex until homogeneous. Process all | + | |
| - | samples before continuing.</br>
| + | |
| - | Notes:
| + | |
| - | <ol style="font-size:large;"> | + | |
| - | <li> | + | |
| - | Combining the RNA Dilution Buffer and Clearing Agent prior to use is not
| + | |
| - | recommended. The Clearing Agent settles more quickly when diluted, and
| + | |
| - | the mixture thickens over time. | + | |
| - | </li> | + | |
| - | <li> | + | |
| - | The Clearing Agent may form a white powder on the threads of the bottle.
| + | |
| - | You can collect the powder with a wet paper towel and discard it as
| + | |
| - | nonhazardous waste.
| + | |
| - | </li> | + | |
| | </ol> | | </ol> |
| - | </li>
| + | |
| - | <li> | + | <h4>Staining with CBB</h4> |
| - | Place the tubes in a heating block or water bath at 70°C. Make sure that the
| + | |
| - | tubes are spaced such that they are fully in contact with the heating block or
| + | |
| - | water and the entire sample is heated. Incubate at 70°C for 5 minutes to
| + | |
| - | denature the samples. The heated mixtures may form clumps of precipitated
| + | |
| - | debris.</br>
| + | |
| - | [!] Incomplete heating of samples may result in DNA contamination.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Remove the tubes and cool the heated samples at room temperature for at
| + | |
| - | least 5 minutes.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Wearing clean gloves, place one blue PureYield™ Clearing Column for each
| + | |
| - | sample in a 50ml collection tube. Save the collection tube caps. Label the Clearing Columns and
| + | |
| - | collection tubes to maintain sample identity</br>
| + | |
| - | Note: If cross-contamination is a concern, flat-top tubes should be used, as
| + | |
| - | they can be sealed during centrifugation.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Mix each sample by vortexing or vigorously shaking until homogeneous.
| + | |
| - | Immediately pour the mixture into the assembled PureYield™ Clearing
| + | |
| - | Column/collection tube. If you are using a flat-top tube, you can seal the
| + | |
| - | assembled column with the cap if desired. Process each sample individually.</br>
| + | |
| - | [!]The flowthrough from the blue PureYield™ Clearing Column must be
| + | |
| - | collected in a collection tube by centrifugation. Vacuum cannot be used.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Centrifuge the PureYield™ Clearing Column assembly in a swinging bucket
| + | |
| - | rotor at 2,000 × g at 22–25°C for 10 minutes to clear the lysate.</br>
| + | |
| - | Note: The Clearing Agent and sample debris will form a solid cake on
| + | |
| - | the surface of the Clearing Membrane. The color may vary, depending on the
| + | |
| - | sample type. If liquid remains on the surface of the debris cake,
| + | |
| - | centrifuge for an additional 5–10 minutes at a higher g force
| + | |
| - | (e.g., 2,500–3,000 × g). A blue-green cleared lysate will collect in the 50ml
| + | |
| - | collection tube.</br>
| + | |
| - | [!]Do not discard the cleared lysate in the collection tube. RNA is present in the
| + | |
| - | blue-green cleared lysate.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Discard the blue Clearing Column, and save the cleared lysate in the
| + | |
| - | collection tube. If a debris pellet is found in the bottom of the collection tube,
| + | |
| - | transfer the cleared lysate to a fresh 50ml tube.
| + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Proceed to RNA Purification by Centrifugation (Spin)
| + | |
| - | </li>
| + | |
| - | </ol>
| + | |
| - | <h4>RNA Purification by Centrifugation (Spin)</h4>
| + | |
| | <ol> | | <ol> |
| - | <li> | + | <li>Put the gel into fixing solution.</li> |
| - | Wearing clean gloves, place a clear PureYield™ Binding Column in a 50ml
| + | <li>Leave the gel to stand with shaking until a band is dyed yellow.</li> |
| - | collection tube for each sample. Save the collection tube caps. Label the
| + | <li>Collect the fixing solution and then put the gel into CBB dyeing liquid.</li> |
| - | columns and tubes to maintain sample identity.</br>
| + | <li>Wrap it and then heat it until it is just before boiling with a microwave oven.</li> |
| - | Note: If cross-contamination is a concern, flat-top tubes should be used, as
| + | <li>Remove the wrap carefully and then let vapor out slowly.</li> |
| - | they can be sealed during centrifugation.
| + | <li>Collect the CBB dyeing liquid and then pour deionized water into a container carrying the gel. Put Kim wipe into the container.</li> |
| - | </li> | + | <li>Infiltrate deionized water into the gel for several tens of minutes. Transfer waste liquid into a tank.</li> |
| - | <li> | + | <li>Take a picture under UV light and then dry the gel and store it.</li> |
| - | Add 4ml isopropanol to the cleared lysate and mix thoroughly by swirling
| + | |
| - | or capping and shaking the tubes. Vortexing is not recommended.
| + | |
| - | Immediately pour the mixture into the PureYield™ Binding
| + | |
| - | Column/collection tube assembly (Step 1). Cap the tube if desired.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Centrifuge at 2,000 × g for 10 minutes in a swinging bucket rotor to capture
| + | |
| - | the RNA. | + | |
| - | </li>
| + | |
| - | <li>
| + | |
| - | Carefully remove the PureYield™ Binding Column from the collection tube
| + | |
| - | and discard the flowthrough. Return the Binding Column to the tube. | + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Verify that ethanol has been added to the RNA Wash Solution. Add 20ml of RNA Wash Solution to the PureYield™ Binding Column. Cap the tube if desired. Centrifuge at 2,000 x g for 5 minutes.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Empty the Collection Tube as in Step 4. Repeat the wash in Step 5 with an
| + | |
| - | additional 10ml of RNA Wash Solution, increasing the centrifugation time
| + | |
| - | to 10 minutes to empty the column and dry the Binding Membrane.</br>
| + | |
| - | Optional: To further reduce ethanol carryover, empty the collection tube
| + | |
| - | and centrifuge the PureYield™ Binding Column for an additional 5
| + | |
| - | minutes at 2,000 × g.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Carefully transfer the PureYield™ Binding Column to a fresh 50ml
| + | |
| - | collection tube. Save the cap for the elution step. Use care to make sure that
| + | |
| - | the flowthrough does not contact the column.</br> | + | |
| - | Note: If the PureYield™ Binding Column is contacted by the flowthrough,
| + | |
| - | empty the collection tube and centrifuge the assembly for 1 minute before
| + | |
| - | transferring the Binding Column to a fresh tube (Step 7).
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Add 1ml Nuclease-Free Water to the PureYield™ Binding Column,
| + | |
| - | preferably using an ART® Aerosol Resistant Tip. Be careful
| + | |
| - | to completely cover the surface of the membrane with the water. If desired,
| + | |
| - | cap the tube with the clean cap. Incubate at room temperature for
| + | |
| - | 2 minutes to release the RNA into solution. Centrifuge at 2,000 x g for
| + | |
| - | 3 minutes to collect the RNA.</br>
| + | |
| - | Note that with some types of centrifuge braking and vacuum systems,
| + | |
| - | some upward displacement of the membrane can occur during the elution
| + | |
| - | step. This does not affect the yield or performance of the system.
| + | |
| - | </li> | + | |
| - | <li> | + | |
| - | Remove the PureYield™ Binding Columns and discard. Store the aliquots
| + | |
| - | of the purified RNA at –70°C using sterile, low-binding (i.e., siliconized),
| + | |
| - | RNase-free microcentrifuge tubes.</br>
| + | |
| - | Note: Ethanol contamination may occur with the centrifugation (spin)
| + | |
| - | format. If the RNA sample is not heat denatured prior to gel loading, it
| + | |
| - | may float out of the well. If this occurs, leave a portion of the sample open
| + | |
| - | to the air for 10 minutes to allow the ethanol to evaporate. The RNA
| + | |
| - | sample may also be incubated at 37°C to increase the evaporation rate.
| + | |
| - | </li> | + | |
| | </ol> | | </ol> |
| | + | <noscript> |
| | <p align="right"><a href="#top">back to page top</a></p> | | <p align="right"><a href="#top">back to page top</a></p> |
| | + | </noscript> |
| | + | |
| | | | |
| - | <h2><a name="WB"></a>Western blotting</h2>
| |
| - | <p align="right"><a href="#top">back to page top</a></p>
| |
| | </div> | | </div> |
| | </div> | | </div> |
| | </body> | | </body> |
| | </html> | | </html> |
"
