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- | <html> | + | <h2>Standard Protocols We Used in the Wet Lab</h2> |
- | <body>
| + | |- |
- | <h1>Protocols: Standard techniques that we used in the wet lab</h1>
| + | |[[Media:Bacterial_Culture.pdf|Bacteria Culture Protocols]] |
- | <h3>Bacterial Culture</h3>
| + | |- |
- | <h4>Sterile Technique</h4>
| + | |[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]] |
- | <ol>
| + | |- |
- | <li>Always work around a flame or in the hood</li>
| + | |[[Media:Plasmid_Prep_from_Cultures.pdf|Plasmid Prep from Cultures]] |
- | <li>Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping</li>
| + | |- |
- | <li>Sterilize metal instruments between uses by dipping in 100% ethanol and flaming</li>
| + | |[[Media:DNA_Quantification.pdf|DNA Quantification]] |
- | </ol>
| + | |- |
- | <br />
| + | |[[Media:PCR.pdf|Polymerase Chain Reaction (PCR)]] |
- | <h4>Bacterial Culture Maintenance</h4>
| + | |- |
- | Culture cells:
| + | |[[Media:Restriction_Digest.pdf|Restriction Digest]] |
- | <ol>
| + | |- |
- | <li>At 36 degrees Celsius</li>
| + | |[[Media:Vector_Dephosphorylation.pdf|Vector Dephosphorylatio]] |
- | <li>Shaking at 220 rpm</li>
| + | |- |
- | <li>At 10% total flask/tube volume</li>
| + | |[[Media:DNA_Fragment_Ligation.pdf|DNA Fragment Ligation]] |
- | <li>In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x10<sup>8</sup>cell/ml)
| + | |- |
- | </ol>
| + | |[[Media:DNA_Purification.pdf|DNA Purification]] |
- | <br />
| + | |- |
- | <h4>Bacterial Culture For Gene Expression Experiments</h4>
| + | |[[Media:Sequencing.pdf|Sequencing]] |
- | <ol>
| + | |- |
- | <li>Pick and individual colony from a plate and inoculate 2ml LB + amp media</li>
| + | |
- | <li>Incubate overnight at 37 C, shaking at 220 rpm</li>
| + | |
- | <li>Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture</li>
| + | |
- | <li>Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)</li>
| + | |
- | <li>Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture</li>
| + | |
- | <li>Continue cultures as described above in "bacterial culture maintenance" for 9 hrs</li>
| + | |
- | <li>Isolate cell samples from cultures at 3, 6, and 9 hour time points</li>
| + | |
- | <li>Remove 100ul sample aliquots from cultures</li>
| + | |
- | <li>Pellet samples at 5K rpm for 5 minutes</li>
| + | |
- | <li>Remove supernatant</li>
| + | |
- | <li>Wash cells with 1 ml chilled 1xPBS, pH 7.6</li>
| + | |
- | <li>Resuspend cells by vortexing</li>
| + | |
- | <li>Re-pellet cells at 5K rpm for 5 minutes</li>
| + | |
- | <li>Remove supernatant</li>
| + | |
- | <li> Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)</li>
| + | |
- | <li>Incubate at RT for 30 minutes</li>
| + | |
- | <li>Pellet cells at 5K rpm for 5 minutes</li>
| + | |
- | <li>Remove supernatant</li>
| + | |
- | <li>Resuspend cells in 1 ml 1xPBS</li>
| + | |
- | <li> Store samples at 4 C until analysis by flow cytometry</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>Transformation of Chemically Competent Cells</h4>
| + | |
- | <ol>
| + | |
- | <li>Thaw cells and incubate transformant DNA in ice(~15 minutes)</li>
| + | |
- | <li>Combine 50 ul cells with ~3uL DNA and mix gently</li>
| + | |
- | <li>Incubate samples on ice for 15 minutes</li>
| + | |
- | <li>Heat shock cells in 42C water bath of 50 seconds</li>
| + | |
- | <li>Incubate samples on ice for 5 minutes</li>
| + | |
- | <li>Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm</li>
| + | |
- | <li>Transfer cells to a 2 ml microfuge tube</li>
| + | |
- | <li>Spin cells down at 6K rpm for 2 minutes</li>
| + | |
- | <li>Remove all but ~100uL supernatant media</li>
| + | |
- | <li>Resuspend cells gently in remaining media</li>
| + | |
- | <li>Plate cells on LB + ab plates</li>
| + | |
- | <li>Incubate plates overnight at 37C</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <br />
| + | |
- | <br />
| + | |
- | <h3>DNA Work</h3>
| + | |
- | <br />
| + | |
- | <h4>Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)</h4>
| + | |
- | <ol>
| + | |
- | <li>Pick and individual colony from a plate and inoculate 2ml LB + ab media</li>
| + | |
- | <li>Incubate culture overnight at 37C</li>
| + | |
- | <li>Transfer culture to 2 ml microfuge tube</li>
| + | |
- | <li>Spin cells down at 13K rpm for 2 min at RT and remove supernatant</li>
| + | |
- | <li>Resuspend cells in 250 ul Buffer P1 (stored at 4 C)</li>
| + | |
- | <li>Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue</li>
| + | |
- | <li>Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless</li>
| + | |
- | <li>Centrifuge sample at 13K rpm for 10 minutes</li>
| + | |
- | <li>Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind</li>
| + | |
- | <li>Centrifugre supernatant into column at 13K rpm for 1 minute</li>
| + | |
- | <li>Remove the flowthrough</li>
| + | |
- | <li>Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute</li>
| + | |
- | <li>Remove the flowthrough</li>
| + | |
- | <li>Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute</li>
| + | |
- | <li>Remove the flowthrough</li>
| + | |
- | <li>Spin out residual liquid at 13K rpm for 1 minute</li>
| + | |
- | <li>Place column in a fresh 1.5 ml microfuge tube</li>
| + | |
- | <li>Elute DNA; apply 40 ul Buffer EB to column, incubate at room temperature for 2 minutes and spin out of column at 13K rpm for one minute</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>DNA quantification</h4>
| + | |
- | <ol>
| + | |
- | <li>Dilute DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul</li>
| + | |
- | <li>Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul</li>
| + | |
- | <li>Read absobance of blank and DNA sample at lambda = 260 and 280</li>
| + | |
- | <li>Calculate [DNA]; [DNA](ng/ul) = DF*A260*50</li>
| + | |
- | <li>Determine sample purity; pure DNA A260/A280 = 1.8</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>Polymerase Chain Reaction(PCR)</h4>
| + | |
- | <ol>
| + | |
- | <li>Combine the following on ice:</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10x Thermo Pol Buffer</td><td>5</td><td>1x</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10mM dNTPs</td><td>1</td><td>0.2mM</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>50mM MgCl<sub>2</sub></td><td>2</td><td>2mM</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10 uM F Primer</td><td>2</td><td>0.4mM</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10 uM R Primer</td><td>2</td><td>0.4uM</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>H<sub>2</sub>O</td><td>Vol req for 50 ul total</td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>DNA</td><td>Vol req for 30 ng <= mass <= 500ng</td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>Taq polymerase</td><td>0.5</td><td>2 U</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td><b>Total</b></td><td><b>50 ul</b></td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Thermocycle</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Segment</th><th>Temperature(C)</th><th>Length<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>1. Initial Denaturation</td><td>94</td><td>3 minutes</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>2. Denaturation</td><td>94</td><td>30 seconds</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>3. Annealing</td><td>~55</td><td>30 seconds</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>4. Extension</td><td>68</td><td>1 min/kb amplified</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>5. Final Extension</td><td>68</td><td>5 minutes</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>6. Final Hold</td><td>12</td><td>Forever</td><td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Repeat 25-35 cycles of segments 2-4</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>Restriction Digest</h4>
| + | |
- | <ol>
| + | |
- | <li>Combine the following on ice:</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10x NEB Buffer</td><td>5</td><td>1x</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>BSA</td><td>0.5</td><td>1x</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>H<sub>2</sub>O</td><td>Vol req for 50 ul total</td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>DNA</td><td>Vol req for 0.5 ng <= mass <= 5 ug</td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>Restriction Enzyme</td><td>0.5</td><td>1 U</td><td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td><b>Total</b></td><td><b>50 ul</b></td><td>-</td><td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Incubate at 37C for 2-20 hours</li>
| + | |
- | <li>Heat inactive the enzyme at 65C for 15 minutes</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>Vector Dephosphorylation</h4>
| + | |
- | <ol>
| + | |
- | <li>Combine the following on ice</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10x AP Buffer</td><td>5</td><td>1x</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>H<sub>2</sub>O</td><td>Vol req for 50 ul</td><td>-</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>DNA</td><td>Vol req for 0.5 ug <= mass <= 5ug</td><td>-</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>Antarctic Phosphate</td><td>0.5</td><td>1 U</td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Incubate at 37C for 30 minutes</li>
| + | |
- | <li>Heat inactive enzyme at 65C for 15 minutes</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>DNA Fragment Ligation</h4>
| + | |
- | <ol>
| + | |
- | <li>Combine the following on ice:</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10x Ligase Buffer</td><td>2</td><td>1x</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>H<sub>2</sub>O</td><td>Vol req for 20 ul</td><td>-</td>
| + | |
- | <tr>
| + | |
- | <td>Vector DNA</td><td>3 <= fmoles <= 30</td><td>-</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>Insert DNA</td><td>9 <= fmoles <=90</td><td>-</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>T4 DNA Ligase</td><td>1</td><td>1 U</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td><b>Total</b></td><td><b>20</b></td><td>-</td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Vector insert ratio should be about 1:3-- lower ratios may decrease insertion efficiency and higher ratios may lead to cancatamerization of inserts. Additionally, 100ng <= total DNA <= 500ng</li>
| + | |
- | <li>Incubate at 16C overnight or at RT for 30 minutes(overnight ligation is preferred)</li>
| + | |
- | <li>Heat inactivate enzyme at 65C for 15 minutes</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <h4>DNA purification</h4>
| + | |
- | <h5>Purify DNA (Using QIAquick PCR purification)</h5>
| + | |
- | <ol>
| + | |
- | <li>Add 5 volumes of Buffer PB1to 1 volume DNA and mix</li>
| + | |
- | <li>Apply DNA sample to QIA quick column</li>
| + | |
- | <li>Spin DNA into column at 13K rpm for 1 minute</li>
| + | |
- | <li> Discard flowthrough</li>
| + | |
- | <li>Wash DNA with 0.75 ml Buffer PE, spin through column at 13K rpm for 1 minute</li>
| + | |
- | <li>Discard flowthrough</li>
| + | |
- | <li>Spin residual liquid from column at 13K rpm for 1 minutes</li>
| + | |
- | <li>Elute DNA; apply 40ul Buffer EB to column, incubate at room temperature for 2 minutes before spinning DNA out of column at 13K rpm for 1 minute</li>
| + | |
- | </ol>
| + | |
- | <h5>Separate DNA by size on an agarose gel</h5>
| + | |
- | <ol>
| + | |
- | <li>Make an agarose gel at 0.8<= gel density <= 1.5</li>
| + | |
- | <li> Add loading dye to samples (5 ul dye/50 ul sample)</li>
| + | |
- | <li>Load samples and a ladder (5 ul) into gel wells</li>
| + | |
- | <li>Run samples through gel (negative to positive) at 100 V for 40 minutes at room temperature</li>
| + | |
- | <li>Visualize DNA under UV light</li>
| + | |
- | </ol>
| + | |
- | <h5>Purify DNA from Gel (Using QIAquick Gel Extraction Kit</h5>
| + | |
- | <ol>
| + | |
- | <li>Excise gel piece containing DNA with a new razor</li>
| + | |
- | <li>Add three volumes of Buffer QG to 1 volume gel</li>
| + | |
- | <li>Incubate at 50C for 15 minutes, or until gel is solublized, mixing frequently</li>
| + | |
- | <li>Make sure dissolved solution is yellow</li>
| + | |
- | <li>If the DNA fragment is <500bp and >4kb, add 1 gel volume isopropanol to increase the yield</li>
| + | |
- | <li>Apply sample to QIAquick spin column in 700 ul aliquots</li>
| + | |
- | <li>Spin sample into column at 13K rpm for 1 minute</li>
| + | |
- | <li>Discard flowthrough</li>
| + | |
- | <li>Spin 0.5 ml Buffer QG through column at 13K rpm for 1 minute to solublize any remaining gel chunks</li>
| + | |
- | <li>Discard flowthrough</li>
| + | |
- | <li>Wash column with 0.75 ml Buffer PE, spinning through column at 13K rpm for 1 minute</li>
| + | |
- | <li>Discard flowthrough</li>
| + | |
- | <li>Spin out residual liquid from column at 13K rpm for 1 minute</li>
| + | |
- | <li>Elute DNA by applying 40-50ul Buffer EB to column, incubate at room temperature for 2 minutes, spin DNA out of the column at 13K rpm for 1 minute into a clean microfuge tube</li>
| + | |
- | </ol>
| + | |
- | <br />
| + | |
- | <br />
| + | |
- | <h3>Sequencing</h3>
| + | |
- | <ol>
| + | |
- | <li>Combine the following in 0.5ml epindorf tube:</li>
| + | |
- | <table>
| + | |
- | <table border="1">
| + | |
- | <tr>
| + | |
- | <th>Reagent</th><th>1x(vol in ul)</th><th>[Final]<th>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>10 uM seq primer</td><td>1</td><td>10 pmol</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>DNA</td><td>Vol req for 300ng</td><td>300 ng</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>H<sub>2</sub>O</td><td>Vol req for 12 uL</td><td>-</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td><b<Total Volume</b></td><td><b>12 ul</b></td><td>-</td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | <li>Submit to BMGC for sequencing</li>
| + | |
- | </ol>
| + | |
- | | + | |
- | | + | |
- | | + | |
| <br /> | | <br /> |
| <br /> | | <br /> |