Team:Lethbridge Canada/protocols
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+ | |||
+ | |||
+ | <div id="mainContent"> | ||
+ | <div id="notebook_header"> | ||
+ | <div id="notebook_links"> | ||
+ | <h1 id="notebook_links_header">Notebook Links</h1> | ||
+ | <ul id="notebook_march_links"> | ||
+ | <li class="notebook_link_title"><a href="notebook_march.html">March</a></li> | ||
+ | <li><a href="notebook_march.html#entry_one">March 12, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_march.html#entry_two">March 13, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
+ | <li><a href="notebook_march.html#entry_three">March 13, 2013: Miniprep and Glycerol Stock</a></li> | ||
+ | <li><a href="notebook_march.html#entry_four">March 15, 2013: Restriction Digestion</a></li> | ||
+ | <li><a href="notebook_march.html#entry_five">March 16, 2013: Agarose Gel</a></li> | ||
+ | <li><a href="notebook_march.html#entry_six">March 18, 2013: Agarose Gel</a></li> | ||
+ | <li><a href="notebook_march.html#entry_seven">March 19, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_march.html#entry_eight">March 20, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
+ | <li><a href="notebook_march.html#entry_nine">March 21, 2013: Miniprep and Glycerol Stock</a></li> | ||
+ | <li><a href="notebook_march.html#entry_ten">March 27, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_march.html#entry_eleven">March 28, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_march.html#entry_twelve">March 28, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
+ | <li><a href="notebook_march.html#entry_thirteen">March 29, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
+ | <li><a href="notebook_march.html#entry_fourteen">March 29, 2013: Picking Cells of -80 Glycerol Stock</a></li> | ||
+ | </ul> | ||
+ | |||
+ | <ul id="notebook_april_links"> | ||
+ | <li class="notebook_link_title"><a href="notebook_april.html">April</a></li> | ||
+ | <li><a href="notebook_april.html#entry_one">April 2, 2013: Restriction - Ligation</a></li> | ||
+ | <li><a href="notebook_april.html#entry_two">April 2, 2013: Ligation</a></li> | ||
+ | <li><a href="notebook_april.html#entry_three">April 3, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_april.html#entry_four">April 4, 2013: Re-Plating Transformation</a></li> | ||
+ | <li><a href="notebook_april.html#entry_five">April 5, 2013: Miniprep of ON Cultures</a></li> | ||
+ | <li><a href="notebook_april.html#entry_six">April 5, 2013: UV Spectroscopy DNA Concentrations</a></li> | ||
+ | <li><a href="notebook_april.html#entry_seven">April 6, 2013: Miniprep From Ligations</a></li> | ||
+ | <li><a href="notebook_april.html#entry_eight">April 7, 2013: PCR</a></li> | ||
+ | <li><a href="notebook_april.html#entry_nine">April 8, 2013: Restriction Digestion of PCR Parts</a></li> | ||
+ | <li><a href="notebook_april.html#entry_ten">April 8, 2013: 3% Agarose Gel of Digested PCR Parts</a></li> | ||
+ | <li><a href="notebook_april.html#entry_eleven">April 8, 2013: Gel Extraction</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twelve">April 9, 2013: Repeat PCR</a></li> | ||
+ | <li><a href="notebook_april.html#entry_thirteen">April 9, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
+ | <li><a href="notebook_april.html#entry_fourteen">April 10, 2013: Miniprep</a></li> | ||
+ | <li><a href="notebook_april.html#entry_fifteen">April 10, 2013: Restriction</a></li> | ||
+ | <li><a href="notebook_april.html#entry_sixteen">April 10, 2013: Agarose Gel of PCR 1%</a></li> | ||
+ | <li><a href="notebook_april.html#entry_seventeen">April 11, 2013: Miniprep</a></li> | ||
+ | <li><a href="notebook_april.html#entry_eighteen">April 12, 2013: Restriction Digestion</a></li> | ||
+ | <li><a href="notebook_april.html#entry_nineteen">April 12, 2013: PCR and Cell Reculturization</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty">April 12, 2013: Miniprep</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_one">April 14, 2013: Miniprep</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_two">April 14, 2013: 1% Agarose Gel</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_six">April 15, 2013: Restriction/Digestion</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_seven">April 15, 2013: Restriction/Digestion</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_three">April 15, 2013: 1% Agarose Gel of Promoters</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_four">April 17, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_april.html#entry_twenty_five">April 30, 2013: 1% Agarose Gel of Promoter Constructs</a></li> | ||
+ | </ul> | ||
+ | |||
+ | <ul id="notebook_may_links"> | ||
+ | <li class="notebook_link_title"><a href="notebook_may.html">May</a></li> | ||
+ | <li><a href="notebook_may.html#entry_one">May 1, 2013: Ligation</a></li> | ||
+ | <li><a href="notebook_may.html#entry_two">May 4, 2013: Restriction Digest</a></li> | ||
+ | <li><a href="notebook_may.html#entry_three">May 4, 2013: 1% Agarose Gel of Promoter Constructs</a></li> | ||
+ | <li><a href="notebook_may.html#entry_four">May 4, 2013: Gel Extractions</a></li> | ||
+ | <li><a href="notebook_may.html#entry_five">May 5, 2013: Transformation</a></li> | ||
+ | <li><a href="notebook_may.html#entry_six">May 7, 2013: Restriction</a></li> | ||
+ | <li><a href="notebook_may.html#entry_seven">May 7, 2013: Gel Extraction</a></li> | ||
+ | <li><a href="notebook_may.html#entry_eight">May 7, 2013: Gel Extracted on Conformation Gel</a></li> | ||
+ | <li><a href="notebook_may.html#entry_nine">May 8, 2013: Ligations</a></li> | ||
+ | <li><a href="notebook_may.html#entry_ten">May 9, 2013: PCR of Ligation Mixture</a></li> | ||
+ | <li><a href="notebook_may.html#entry_eleven">May 13, 2013: Restriction of PCR Ligations Gel Conformation</a></li> | ||
+ | <li><a href="notebook_may.html#entry_twelve">May 14, 2013: Transformation of Ligations</a></li> | ||
+ | <li><a href="notebook_may.html#entry_thirteen">May 15, 2013: Ligation</a></li> | ||
+ | <li><a href="notebook_may.html#entry_fourteen">May 15, 2013: Picking Cells</a></li> | ||
+ | <li><a href="notebook_may.html#entry_fifteen">May 16, 2013: Picking Cells</a></li> | ||
+ | <li><a href="notebook_may.html#entry_sixteen">May 16, 2013: 10 Fold Miniprep</a></li> | ||
+ | <li><a href="notebook_may.html#entry_seventeen">May 17, 2013: Agarose Gel</a></li> | ||
+ | <li><a href="notebook_may.html#entry_eighteen">May 17, 2013: Miniprep</a></li> | ||
+ | <li><a href="notebook_may.html#entry_nineteen">May 17, 2013: Glycerol Stock</a></li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <h1 id="protocol_header">Protocols:</h1> | ||
+ | <div id="protocols_content"> | ||
+ | <div class="protocol_entry"> | ||
+ | <div id="agarose"> | ||
+ | <h1>Agarose Gel Electrophoresis</h1> | ||
+ | |||
+ | <h2>Prepare the Agarose Gel:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Weigh appropriate amount of agarose into a small Erlenmeyer flask (0.3g for 30mL small 1% gel).</li> | ||
+ | <li>Add desired volume of 1x TAE buffer (or 1x TBE in special cases) (30mL for small gel chamber).</li> | ||
+ | <li>Weight the flask and write down its total weight.</li> | ||
+ | <li>Boil it to resolve the agarose: | ||
+ | <ul> | ||
+ | <li>either by microwaving (without magnetic stir bar)</li> | ||
+ | <li>or on hot plate (with magnetic stir bar).</li> | ||
+ | </ul> | ||
+ | <li>Let it cool down while stirring to about 60°C (hand-warm).</li> | ||
+ | <li>Weight again and replace lost water.</li> | ||
+ | <li>Seal ends of gel plate with black wedges.</li> | ||
+ | <li>Cast gel into gel plate and put in the comb, wait until solid.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Prepare Samples:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Transfer about 0.3μg DNA into a microcentrifuge tube.</li> | ||
+ | <li>Add 1x TAE to 5μL.</li> | ||
+ | <li>Add 1μL of 6x DNA sample buffer.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Run Electrophoresis:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Place gel into the big chamber in correct orientation (DNA migrates to the positive pole)</li> | ||
+ | <li>Fill chamber with 1x TAE buffer (or 1x TBE), about 0.5cm above gel.</li> | ||
+ | <li>Carefully remove comb from solid gel.</li> | ||
+ | <li>Load the 6 μL samples into the gel slots, and load 4 μL of DNA ladder (typically 1kb ladder) in at least one gel slot.</li> | ||
+ | <li>Close chamber with lid in the correct orientation (black/red).</li> | ||
+ | <li>Connect cables to power supply.</li> | ||
+ | <li>Run the gel with 1–5V/cm of gel length, check for dye fronts should be at about 1/3 and 2/3 of gels (about 1h) – common lab procedure: 100V for small gel.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Staining and Photo:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Stain the gels for 10–20 minutes in ethidiumbromide while shaking. Attention: always wear gloves with ethidiumbromide, try not to touch the solution or the stained gel but use a kitchen spatula to handle the gel.</li> | ||
+ | <li>Destain the gel for a short while in water (e.g. while transporting it to take the photo).</li> | ||
+ | <li>Observe bands on UV illuminator and take a digital photo. Carfeful: strong UV light, wear glasses and lab coat to avoid “sun burn."</li> | ||
+ | <li>Clean UV illuminator.</li> | ||
+ | <li>Discard gel in ethidiumbromide waste.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Buffers:</h2> | ||
+ | |||
+ | <div id="agarose_buffers"> | ||
+ | |||
+ | <div class="border_right"> | ||
+ | <h3>50x TAE - Dilution to 4L of 1x TAE Buffer:</h3> | ||
+ | <ul> | ||
+ | <li>242g Tris </li> | ||
+ | <li>80ml 50x TAE buffer</li> | ||
+ | <li>57.1mL acetic acid </li> | ||
+ | <li>fill up to 4L with MilliQ H<sub>2</sub>O</li> | ||
+ | <li>100mL 0.5 M EDTA pH 8.0</li> | ||
+ | <li>H<sub>2</sub>O to final volume of 1L</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | <div class="border_right"> | ||
+ | <h3>55x TBE:</h3> | ||
+ | <ul> | ||
+ | <li>54g Tris base - end concentration 90mM</li> | ||
+ | <li>27.5mL boric acid - end concentration 90mM</li> | ||
+ | <li>20 ml 0.5 M EDTA ph 8.0 - end concentration 1mM</li> | ||
+ | <li>H<sub>2</sub>O to final volume of 1L | ||
+ | <ul> | ||
+ | <li>Should be pH 8.3 without adjustment</li> | ||
+ | <li>Filter to delay precipitation</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | <h3>6x DNA Loading Buffer:</h3> | ||
+ | <ul> | ||
+ | <li>0.25% bromphenol blue</li> | ||
+ | <li>0.25% Xylencyanol</li> | ||
+ | <li>60% Glycerol</li> | ||
+ | <li>1% SDS</li> | ||
+ | <li>20mM EDTA</li> | ||
+ | <li>In H<sub>2</sub>O</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <h2>Gene Ruler 1kb DNA ladder (Fermentas)</h2> | ||
+ | |||
+ | <table class="protocols_table"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td rowspan="4">1:6 Dilution</td> | ||
+ | </tr> | ||
+ | <tr><td>100μLDNA ladder (0.5 μg/μL)</td></tr> | ||
+ | <tr><td>100μL6x Loading Dye</td></tr> | ||
+ | <tr><td>400μMilliQ H<sub>2</sub>O</td></tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Results in 0.08333μg/μL, i.e. 0.5μg in 6μL as recommended by Fermentas</p> | ||
+ | |||
+ | <h2>Gel Concentration to Separate Various Sizes of DNA:</h2> | ||
+ | |||
+ | <table class="protocols_table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Agarose in % (w/v)</th> | ||
+ | <th>Optimal for Linear Double-Stranded DNA (size in kb)</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>0.3</td> | ||
+ | <td>5 - 60</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>0.6</td> | ||
+ | <td>1 - 20</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>0.7</td> | ||
+ | <td>0.8 - 10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>0.9</td> | ||
+ | <td>0.5 - 7</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1.2</td> | ||
+ | <td>0.4 - 6</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1.5</td> | ||
+ | <td>0.2 - 3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.0</td> | ||
+ | <td>0.1 - 2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3.0*</td> | ||
+ | <td>optimal for 50–100bp fragments, e.g. templates for in vitro transcription</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p class="italic">* For gels of more than 2%, use 3 parts “cheap” agarose and 1 part Top Vision Agarose, e.g. 0.9g cheap agarose + 0.3g Top Vision (not low melting) agarose for 40mL of 3.% gel</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Agarose Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Overnight Cultures Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | |||
+ | <div class="protocol_entry"> | ||
+ | <div id="overnight_cultures"> | ||
+ | |||
+ | <h1>Overnight Cultures</h1> | ||
+ | |||
+ | <h2>Overnight Culture of Bacteria for Plasmid Purification:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Using proper Aseptic technique, sterilely transfer 5mL of LB medium into a sterile culture tube or falcon tube.</li> | ||
+ | <li>Thaw the appropriate antibiotic and pipette in the amount needed.</li> | ||
+ | <li>Sterilize the inoculating loop using flame and allow cooling then carefully picking one colony from a plate or scraping some media from a glycerol stock and transferring the cells to the media by swirling the inoculating loop.</li> | ||
+ | <li>Secure the lids of the vessel and incubate overnight with shaking at appropriate temp (usually 37°C)</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Plasmid Purification:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>If a glycerol stock of the bacteria has not previously been made do so now by cutting the end off of a 1000μL tip and pipette 200μL of 100% autoclaved glycerol into a cryomicrofuge tube. | ||
+ | <ul> | ||
+ | <li>Glycerol is very viscus and this will take practice to transfer 200μL accurately.</li> | ||
+ | <li>Do not pipet from the common glycerol bottle! Always pour some glycerol into a microfuge tube and keep your stock separate</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Pipette 800μLo f overnight culture into the cryovial and mix by swirling the tip and carefully pipetting up and down. Be careful not to suck any media into the barrel of you pipette.</li> | ||
+ | <li>Freeze the cells in liquid nitrogen and store in the -800°C freezer</li> | ||
+ | <li>Transfer the remaining bacteria into a falcon tube and centrifuge at 5000XG for 7 minutes. Alternately you can pipette cells 1.5 mL at a time in a microfuge tube at max speed for 1.5 minutes.</li> | ||
+ | <li>Remove the supernatant and dispose of in the bacteria waste vessel and proceed to the miniprep protocol found in the miniprep kits.</li> | ||
+ | </ol> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Overnight Cultures Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Restriction Digestion Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | |||
+ | <div class="protocol_entry"> | ||
+ | <div id="restriction_digestion"> | ||
+ | |||
+ | <h1>Restriction Digestion</h1> | ||
+ | |||
+ | <ol> | ||
+ | <li>Pipet in this order into a 1.5ml microcentrifuge tube: | ||
+ | <ol> | ||
+ | <li>MilliQ H<sub>2</sub>O - final total volume: 20μL</li> | ||
+ | <li>10x Buffer (corresponding to enzyme) - 2μL</li> | ||
+ | <li>Plasmid DNA (volume according to concentration) 2μg(end concentration < 0.3μg/μL)</li> | ||
+ | <li>Restriction enzyme - 1-2U/μg DNA | ||
+ | <ul> | ||
+ | <li>(Volume according to concentration stated on enzyme tube)</li> | ||
+ | <li>(Don’t pipet less than 0.25μL)</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Attention with handling restriction enzymes: take them only immediately before use out of -20°C fridge, store on ice, put back into fridge as soon as possible.</li> | ||
+ | </ol> | ||
+ | </li> | ||
+ | <li>Incubate at optimal for 1 hour (some enzymes need longer times E.g. SalI) (incubation at 37°C in water bath, but e.g. for SmaI 25-30°C).</li> | ||
+ | <li>Take sample for agarose gel or store for short-term on ice or for long-term at -20°C.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Preparative:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Parts: | ||
+ | <ul> | ||
+ | <li>MilliQ - 30μL</li> | ||
+ | <li>10x Buffer (corresponding to enzyme) - 1/10 of final volume 3μL</li> | ||
+ | <li>Plasmid DNA - 0.2μg/μL, 6μg</li> | ||
+ | <li>Restriction enzyme - 1U/μg DNA</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Incubate 2 hours at 37°C</li> | ||
+ | <li>Note: | ||
+ | <ul> | ||
+ | <li>SmaI needs to be incubated at 25-30°C (only 50% activity at 37°C)</li> | ||
+ | <li>SalI needs long incubation times (>4h)</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ol> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Restriction Digestion Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Miniprep Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | |||
+ | |||
+ | <div class="protocol_entry"> | ||
+ | <div id="miniprep"> | ||
+ | |||
+ | <h1>Miniprep Using EZ-10 Spin Column Kits (High Copy Number Plasmid)</h1> | ||
+ | |||
+ | <h2>Procedure:</h2> | ||
+ | |||
+ | <ol> | ||
+ | <li>Warm Elution Buffer in incubator or in water bath at 37-50°C.</li> | ||
+ | <li>Add 1.5mL of the overnight culture to a 1.5mL microcentrifuge tube and centrifuge at 12000rpm for 2 minutes. Drain supernatant and repeat until all of the overnight culture is used.</li> | ||
+ | <li>Add 100μL of Solution 1 to the cell pellet, mix by pipetting, let stand 1 minute.</li> | ||
+ | <li>Add 200μL of Solution 2 to the mixture, mix gently by inverting the tube 4-6 times and let stand for 1 minute. Do not vortex.</li> | ||
+ | <li>Add 350μL of Solution 3 and mix gently by inverting the tube 4-6 times and let stand for 1 minute.</li> | ||
+ | <li>Centrifuge at 12000rpm for 5 minutes.</li> | ||
+ | <li>Transfer supernatant to an EZ-10 Spin Column and centrifuge at 10000 rpm for 2 minutes.</li> | ||
+ | <li>Discard the flow through in the tube. Add 500μL of Wash Solution to the column and centrifuge at 10000 rpm for 2 minutes.</li> | ||
+ | <li>Repeat wash procedure in Step 7.</li> | ||
+ | <li>Discard the flow though in the collection tube. Centrifuge at 10000rpm for an additional minute to remove any residual wash solution.</li> | ||
+ | <li>Transfer the column to a clean, labelled 1.5 mL microcentrifuge tube. Add 50μL of Elution Buffer to the centre part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10000 rpm for 2 minutes.</li> | ||
+ | <li>Store purified DNA at -20°C.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Labeling:</h2> | ||
+ | |||
+ | <p>All tubes are to be labeled with this information:</p> | ||
+ | |||
+ | <ul> | ||
+ | <li>Name</li> | ||
+ | <li>Date</li> | ||
+ | <li>Antibiotic</li> | ||
+ | <li>Species</li> | ||
+ | <li>Strain</li> | ||
+ | <li>Part Number and Plasmid</li> | ||
+ | </ul> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Miniprep Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Transformation Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | <div class="protocol_entry"> | ||
+ | <div id="transformation"> | ||
+ | |||
+ | <h1>Transformation of Competent Cells</h1> | ||
+ | |||
+ | <ol> | ||
+ | <li>Thaw 20 μL of pre aliquotted cells (Dh5α or BL21 DE3) on ice. Competent cells are stored at -80°C. (Often cells are frozen as 50 μL aliquots – split under sterile conditions for 2 transformations.)</li> | ||
+ | <li>Gently pipet maximum 2.0μL (better 1.8μL) of DNA into the competent cells and pipet once up and down to rinse the tip. | ||
+ | <ul> | ||
+ | <li>Never use more DNA than 10% of the volume of the competent cells, otherwise the cells get destroyed by osmotic shock.</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Mix the DNA into the cells by swirling the tip in the solution.</li> | ||
+ | <li>Incubate the cells on ice for 30 minutes.</li> | ||
+ | <li>Heat shock the cells in a water bath at 42°C for exactly 45 seconds.</li> | ||
+ | <li>Incubate the cells on ice 1 minute.</li> | ||
+ | <li>Add 250 μL of sterile media to the cells and incubate at 37°C for 1 hour with shaking (tape microcentrifuge tube in shaking incubator).</li> | ||
+ | <li>Label the LB plates on the outside perimeter: | ||
+ | <ol id="trans_labeling"> | ||
+ | <li>Your name</li> | ||
+ | <li>Date</li> | ||
+ | <li>Cell strain (e.g. DH5α, BL21DE3 etc.)</li> | ||
+ | <li>Plasmid</li> | ||
+ | <li>Volume plated</li> | ||
+ | </ol> | ||
+ | </li> | ||
+ | <li>Plate 100 μL and 50 μL on pre-warmed LB plates containing the appropriate antibiotic. For ligations and mutagenesis: plate all 250 μL on (1 or) 2 plate.</li> | ||
+ | <li>Leave plate for 10-15 minutes to soak the cell suspension into the agar.</li> | ||
+ | <li>Flip plate over (agar on top).</li> | ||
+ | <li>Incubate the plates in the 37°C oven overnight.</li> | ||
+ | <li>Keep the remaining solution in the 4°C fridge overnight until transformation has been confirmed.</li> | ||
+ | </ol> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Transformation Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| --> | ||
+ | </div> | ||
+ | </div> | ||
+ | </body> | ||
+ | </html> |
Revision as of 23:37, 6 June 2013
Notebook Links
- March
- March 12, 2013: Transformation
- March 13, 2013: Picking Cell Colonies (Cultures)
- March 13, 2013: Miniprep and Glycerol Stock
- March 15, 2013: Restriction Digestion
- March 16, 2013: Agarose Gel
- March 18, 2013: Agarose Gel
- March 19, 2013: Transformation
- March 20, 2013: Picking Cell Colonies (Cultures)
- March 21, 2013: Miniprep and Glycerol Stock
- March 27, 2013: Transformation
- March 28, 2013: Transformation
- March 28, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cells of -80 Glycerol Stock
- April
- April 2, 2013: Restriction - Ligation
- April 2, 2013: Ligation
- April 3, 2013: Transformation
- April 4, 2013: Re-Plating Transformation
- April 5, 2013: Miniprep of ON Cultures
- April 5, 2013: UV Spectroscopy DNA Concentrations
- April 6, 2013: Miniprep From Ligations
- April 7, 2013: PCR
- April 8, 2013: Restriction Digestion of PCR Parts
- April 8, 2013: 3% Agarose Gel of Digested PCR Parts
- April 8, 2013: Gel Extraction
- April 9, 2013: Repeat PCR
- April 9, 2013: Picking Cell Colonies (Cultures)
- April 10, 2013: Miniprep
- April 10, 2013: Restriction
- April 10, 2013: Agarose Gel of PCR 1%
- April 11, 2013: Miniprep
- April 12, 2013: Restriction Digestion
- April 12, 2013: PCR and Cell Reculturization
- April 12, 2013: Miniprep
- April 14, 2013: Miniprep
- April 14, 2013: 1% Agarose Gel
- April 15, 2013: Restriction/Digestion
- April 15, 2013: Restriction/Digestion
- April 15, 2013: 1% Agarose Gel of Promoters
- April 17, 2013: Transformation
- April 30, 2013: 1% Agarose Gel of Promoter Constructs
- May
- May 1, 2013: Ligation
- May 4, 2013: Restriction Digest
- May 4, 2013: 1% Agarose Gel of Promoter Constructs
- May 4, 2013: Gel Extractions
- May 5, 2013: Transformation
- May 7, 2013: Restriction
- May 7, 2013: Gel Extraction
- May 7, 2013: Gel Extracted on Conformation Gel
- May 8, 2013: Ligations
- May 9, 2013: PCR of Ligation Mixture
- May 13, 2013: Restriction of PCR Ligations Gel Conformation
- May 14, 2013: Transformation of Ligations
- May 15, 2013: Ligation
- May 15, 2013: Picking Cells
- May 16, 2013: Picking Cells
- May 16, 2013: 10 Fold Miniprep
- May 17, 2013: Agarose Gel
- May 17, 2013: Miniprep
- May 17, 2013: Glycerol Stock
Protocols:
Agarose Gel Electrophoresis
Prepare the Agarose Gel:
- Weigh appropriate amount of agarose into a small Erlenmeyer flask (0.3g for 30mL small 1% gel).
- Add desired volume of 1x TAE buffer (or 1x TBE in special cases) (30mL for small gel chamber).
- Weight the flask and write down its total weight.
- Boil it to resolve the agarose:
- either by microwaving (without magnetic stir bar)
- or on hot plate (with magnetic stir bar).
- Let it cool down while stirring to about 60°C (hand-warm).
- Weight again and replace lost water.
- Seal ends of gel plate with black wedges.
- Cast gel into gel plate and put in the comb, wait until solid.
Prepare Samples:
- Transfer about 0.3μg DNA into a microcentrifuge tube.
- Add 1x TAE to 5μL.
- Add 1μL of 6x DNA sample buffer.
Run Electrophoresis:
- Place gel into the big chamber in correct orientation (DNA migrates to the positive pole)
- Fill chamber with 1x TAE buffer (or 1x TBE), about 0.5cm above gel.
- Carefully remove comb from solid gel.
- Load the 6 μL samples into the gel slots, and load 4 μL of DNA ladder (typically 1kb ladder) in at least one gel slot.
- Close chamber with lid in the correct orientation (black/red).
- Connect cables to power supply.
- Run the gel with 1–5V/cm of gel length, check for dye fronts should be at about 1/3 and 2/3 of gels (about 1h) – common lab procedure: 100V for small gel.
Staining and Photo:
- Stain the gels for 10–20 minutes in ethidiumbromide while shaking. Attention: always wear gloves with ethidiumbromide, try not to touch the solution or the stained gel but use a kitchen spatula to handle the gel.
- Destain the gel for a short while in water (e.g. while transporting it to take the photo).
- Observe bands on UV illuminator and take a digital photo. Carfeful: strong UV light, wear glasses and lab coat to avoid “sun burn."
- Clean UV illuminator.
- Discard gel in ethidiumbromide waste.
Buffers:
50x TAE - Dilution to 4L of 1x TAE Buffer:
- 242g Tris
- 80ml 50x TAE buffer
- 57.1mL acetic acid
- fill up to 4L with MilliQ H2O
- 100mL 0.5 M EDTA pH 8.0
- H2O to final volume of 1L
55x TBE:
- 54g Tris base - end concentration 90mM
- 27.5mL boric acid - end concentration 90mM
- 20 ml 0.5 M EDTA ph 8.0 - end concentration 1mM
- H2O to final volume of 1L
- Should be pH 8.3 without adjustment
- Filter to delay precipitation
6x DNA Loading Buffer:
- 0.25% bromphenol blue
- 0.25% Xylencyanol
- 60% Glycerol
- 1% SDS
- 20mM EDTA
- In H2O
Gene Ruler 1kb DNA ladder (Fermentas)
1:6 Dilution |
100μLDNA ladder (0.5 μg/μL) |
100μL6x Loading Dye |
400μMilliQ H2O |
Results in 0.08333μg/μL, i.e. 0.5μg in 6μL as recommended by Fermentas
Gel Concentration to Separate Various Sizes of DNA:
Agarose in % (w/v) | Optimal for Linear Double-Stranded DNA (size in kb) |
---|---|
0.3 | 5 - 60 |
0.6 | 1 - 20 |
0.7 | 0.8 - 10 |
0.9 | 0.5 - 7 |
1.2 | 0.4 - 6 |
1.5 | 0.2 - 3 |
2.0 | 0.1 - 2 |
3.0* | optimal for 50–100bp fragments, e.g. templates for in vitro transcription |
* For gels of more than 2%, use 3 parts “cheap” agarose and 1 part Top Vision Agarose, e.g. 0.9g cheap agarose + 0.3g Top Vision (not low melting) agarose for 40mL of 3.% gel
Overnight Cultures
Overnight Culture of Bacteria for Plasmid Purification:
- Using proper Aseptic technique, sterilely transfer 5mL of LB medium into a sterile culture tube or falcon tube.
- Thaw the appropriate antibiotic and pipette in the amount needed.
- Sterilize the inoculating loop using flame and allow cooling then carefully picking one colony from a plate or scraping some media from a glycerol stock and transferring the cells to the media by swirling the inoculating loop.
- Secure the lids of the vessel and incubate overnight with shaking at appropriate temp (usually 37°C)
Plasmid Purification:
- If a glycerol stock of the bacteria has not previously been made do so now by cutting the end off of a 1000μL tip and pipette 200μL of 100% autoclaved glycerol into a cryomicrofuge tube.
- Glycerol is very viscus and this will take practice to transfer 200μL accurately.
- Do not pipet from the common glycerol bottle! Always pour some glycerol into a microfuge tube and keep your stock separate
- Pipette 800μLo f overnight culture into the cryovial and mix by swirling the tip and carefully pipetting up and down. Be careful not to suck any media into the barrel of you pipette.
- Freeze the cells in liquid nitrogen and store in the -800°C freezer
- Transfer the remaining bacteria into a falcon tube and centrifuge at 5000XG for 7 minutes. Alternately you can pipette cells 1.5 mL at a time in a microfuge tube at max speed for 1.5 minutes.
- Remove the supernatant and dispose of in the bacteria waste vessel and proceed to the miniprep protocol found in the miniprep kits.
Restriction Digestion
- Pipet in this order into a 1.5ml microcentrifuge tube:
- MilliQ H2O - final total volume: 20μL
- 10x Buffer (corresponding to enzyme) - 2μL
- Plasmid DNA (volume according to concentration) 2μg(end concentration < 0.3μg/μL)
- Restriction enzyme - 1-2U/μg DNA
- (Volume according to concentration stated on enzyme tube)
- (Don’t pipet less than 0.25μL)
- Attention with handling restriction enzymes: take them only immediately before use out of -20°C fridge, store on ice, put back into fridge as soon as possible.
- Incubate at optimal for 1 hour (some enzymes need longer times E.g. SalI) (incubation at 37°C in water bath, but e.g. for SmaI 25-30°C).
- Take sample for agarose gel or store for short-term on ice or for long-term at -20°C.
Preparative:
- Parts:
- MilliQ - 30μL
- 10x Buffer (corresponding to enzyme) - 1/10 of final volume 3μL
- Plasmid DNA - 0.2μg/μL, 6μg
- Restriction enzyme - 1U/μg DNA
- Incubate 2 hours at 37°C
- Note:
- SmaI needs to be incubated at 25-30°C (only 50% activity at 37°C)
- SalI needs long incubation times (>4h)
Miniprep Using EZ-10 Spin Column Kits (High Copy Number Plasmid)
Procedure:
- Warm Elution Buffer in incubator or in water bath at 37-50°C.
- Add 1.5mL of the overnight culture to a 1.5mL microcentrifuge tube and centrifuge at 12000rpm for 2 minutes. Drain supernatant and repeat until all of the overnight culture is used.
- Add 100μL of Solution 1 to the cell pellet, mix by pipetting, let stand 1 minute.
- Add 200μL of Solution 2 to the mixture, mix gently by inverting the tube 4-6 times and let stand for 1 minute. Do not vortex.
- Add 350μL of Solution 3 and mix gently by inverting the tube 4-6 times and let stand for 1 minute.
- Centrifuge at 12000rpm for 5 minutes.
- Transfer supernatant to an EZ-10 Spin Column and centrifuge at 10000 rpm for 2 minutes.
- Discard the flow through in the tube. Add 500μL of Wash Solution to the column and centrifuge at 10000 rpm for 2 minutes.
- Repeat wash procedure in Step 7.
- Discard the flow though in the collection tube. Centrifuge at 10000rpm for an additional minute to remove any residual wash solution.
- Transfer the column to a clean, labelled 1.5 mL microcentrifuge tube. Add 50μL of Elution Buffer to the centre part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10000 rpm for 2 minutes.
- Store purified DNA at -20°C.
Labeling:
All tubes are to be labeled with this information:
- Name
- Date
- Antibiotic
- Species
- Strain
- Part Number and Plasmid
Transformation of Competent Cells
- Thaw 20 μL of pre aliquotted cells (Dh5α or BL21 DE3) on ice. Competent cells are stored at -80°C. (Often cells are frozen as 50 μL aliquots – split under sterile conditions for 2 transformations.)
- Gently pipet maximum 2.0μL (better 1.8μL) of DNA into the competent cells and pipet once up and down to rinse the tip.
- Never use more DNA than 10% of the volume of the competent cells, otherwise the cells get destroyed by osmotic shock.
- Mix the DNA into the cells by swirling the tip in the solution.
- Incubate the cells on ice for 30 minutes.
- Heat shock the cells in a water bath at 42°C for exactly 45 seconds.
- Incubate the cells on ice 1 minute.
- Add 250 μL of sterile media to the cells and incubate at 37°C for 1 hour with shaking (tape microcentrifuge tube in shaking incubator).
- Label the LB plates on the outside perimeter:
- Your name
- Date
- Cell strain (e.g. DH5α, BL21DE3 etc.)
- Plasmid
- Volume plated
- Plate 100 μL and 50 μL on pre-warmed LB plates containing the appropriate antibiotic. For ligations and mutagenesis: plate all 250 μL on (1 or) 2 plate.
- Leave plate for 10-15 minutes to soak the cell suspension into the agar.
- Flip plate over (agar on top).
- Incubate the plates in the 37°C oven overnight.
- Keep the remaining solution in the 4°C fridge overnight until transformation has been confirmed.