Team:WashU StLouis/Protocol
From 2014.igem.org
(Difference between revisions)
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<table id="general"> | <table id="general"> | ||
<tr> <td> | <tr> <td> | ||
- | <center><h1> Protocol </h1> | + | <center><h1> Protocol </h1> |
<p> Because Jeffrey & Ben (Brauer), and Richard & Caroline (Rebstock) are working in two separate labs, the different groups have their own set of protocols that they adhere by. Listed below are the protocols they used for cloning:</p> </center> | <p> Because Jeffrey & Ben (Brauer), and Richard & Caroline (Rebstock) are working in two separate labs, the different groups have their own set of protocols that they adhere by. Listed below are the protocols they used for cloning:</p> </center> | ||
</td> | </td> | ||
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<table id="general"> | <table id="general"> | ||
<tr> <td> | <tr> <td> | ||
- | <h1> Brauer Group Protocols </h1> | + | <center><h1> Brauer Group Protocols </h1></center> |
<a name="1"></a> | <a name="1"></a> | ||
+ | |||
+ | <p><h3>Polymerase Chain Reaction</h3></p> | ||
+ | |||
+ | <p>Phusion HF Polymerase Protocol</p> | ||
+ | |||
+ | <p>Per tube, add in this order:</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>Cloning water</p></td> | ||
+ | <td valign="top"><p>21.5 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>5x HF buffer</p></td> | ||
+ | <td valign="top"><p>10 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>5M Butane</p></td> | ||
+ | <td valign="top"><p>10 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>DMSO</p></td> | ||
+ | <td valign="top"><p>4 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>10µM Primers (f+r)</p></td> | ||
+ | <td valign="top"><p>2.5 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>10µM dNTPs</p></td> | ||
+ | <td valign="top"><p>1.0 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Template DNA</p></td> | ||
+ | <td valign="top"><p>0.5 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Phusion Polymerase</p></td> | ||
+ | <td valign="top"><p>0.5 µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>PCR Reaction in Thermal Cycler</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>98 °C</p></td> | ||
+ | <td valign="top"><p>30 seconds</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>30 Cycles</p></td> | ||
+ | <td valign="top"> <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>98 °C</p></td> | ||
+ | <td valign="top"><p>20 seconds</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Annealing Temperature (NEB TM Calculator)</p></td> | ||
+ | <td valign="top"><p>20 seconds</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>72 °C</p></td> | ||
+ | <td valign="top"><p>Extension Time (20 seconds/kb)</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>72 °C</p></td> | ||
+ | <td valign="top"><p>5 minutes</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>4 °C</p></td> | ||
+ | <td valign="top"><p>Forever</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Colony PCR</h3></p> | ||
+ | |||
+ | <p>1. Prepare 10 µl of cloning water per colony to be picked in PCR tubes.</p> | ||
+ | |||
+ | <p>2. Prepare culture tube filled with 4mL LB, 4µL resistance</p> | ||
+ | |||
+ | <p>3. Pick colony and resuspend in cloning water by pipetting up and down</p> | ||
+ | |||
+ | <p>4. Next, drop the tip inside the culture tube, or dot onto pre-numbered LB plate</p> | ||
+ | |||
+ | <p>5. Heat water with colonies for 10 minutes in 98°C; use as “template DNA” </p> | ||
+ | |||
+ | <p>6. Put culture tubes in 37°C shaker to incubate (overnight) while PCR is running.</p> | ||
+ | |||
+ | <p>7. Go TAQ PCR mix</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>Cloning Water</p></td> | ||
+ | <td valign="top"><p>35µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Vortexed 5x Green Taq Buffer</p></td> | ||
+ | <td valign="top"><p>10µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>10µM Sequencing Primer</p></td> | ||
+ | <td valign="top"><p>2.5µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>10µM dNTPs</p></td> | ||
+ | <td valign="top"><p>1.0µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Template DNA (heated above)</p></td> | ||
+ | <td valign="top"><p>1.0µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>GoTaq Polymerase</p></td> | ||
+ | <td valign="top"><p>0.5µl</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>8. TAQ PCR Protocol</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>95 °C</p></td> | ||
+ | <td valign="top"><p>2 minutes</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>35 Cycles</p></td> | ||
+ | <td valign="top"> <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>95 °C</p></td> | ||
+ | <td valign="top"><p>1 minute</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>Annealing Temperature (NEB TM Calculator)</p></td> | ||
+ | <td valign="top"><p>45 seconds</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>72 °C</p></td> | ||
+ | <td valign="top"><p>Extension Time (20 seconds/kb)</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>72 °C</p></td> | ||
+ | <td valign="top"><p>5 minutes</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>4 °C</p></td> | ||
+ | <td valign="top"><p>Forever</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>9. Run Gel Visualization</p> | ||
+ | |||
+ | <p>10. Add 15µl PCR product (no need 6x loading dye)</p> | ||
+ | |||
+ | <p>11. Freeze & Miniprep cultures that have desired bands</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Gradient PCR</h3></p> | ||
+ | |||
+ | <p>Annealing Temperature: 5 °C below to 5 °C above. </p> | ||
+ | |||
+ | <p>Example: 62°C = 57°C -> 67°C</p> | ||
+ | |||
+ | <p>Increase Annealing Time to 30 seconds</p> | ||
+ | |||
+ | <p><h3><br /> | ||
+ | </h3></p> | ||
+ | |||
+ | <p><h3>Making a Gel for Gel Electrophoresis</h3></p> | ||
+ | |||
+ | <p>1. 0.7g of Agarose</p> | ||
+ | |||
+ | <p>2. Add 100 ml 1x TAE buffer into an Erlenmeyer flask</p> | ||
+ | |||
+ | <p>3. Microwave for 2 minutes</p> | ||
+ | |||
+ | <p>4. Cool down under tap water for 20 seconds</p> | ||
+ | |||
+ | <p>5. Add 5 µl Sybrsafe</p> | ||
+ | |||
+ | <p>6. Pour the gel into mold with comb. Let cool for 30 minutes.</p> | ||
+ | |||
+ | <p>7. Remove the comb. </p> | ||
+ | |||
+ | <p>8. Transfer tray into an electrophoresis chamber, wells facing black side (anode) of chamber</p> | ||
+ | |||
+ | <p>9. Fill chamber with 1x TAE until TAE just covers the gel</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Gel visualization/extraction</h3></p> | ||
+ | |||
+ | <p>1. Prepare samples</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>Visualization</p></td> | ||
+ | <td valign="top"><p>Extraction</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>10µl PCR Product</p></td> | ||
+ | <td valign="top"><p>50µl PCR Product</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>2µl 6x Loading Dye</p></td> | ||
+ | <td valign="top"><p>10µl 6x Loading Dye</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>2. Add DNA ladder Standard into first well.</p> | ||
+ | |||
+ | <p>3. Pipet prepared samples into rest of wells.</p> | ||
+ | |||
+ | <p>4. Seal electrophoresis chamber with chamber cap and plug cables in VWR power unit.</p> | ||
+ | |||
+ | <p>5. Set 125V, Run for 30 minutes (or longer) depending on length of sample expected.</p> | ||
+ | |||
+ | <p>6. Remove Gel tray once finished and visualize using trans-illuminator. </p> | ||
+ | |||
+ | <p>7. Extract if necessary into a preweighed 1.5 ml microcentrifuge tube.</p> | ||
+ | |||
+ | <p><h3><br /> | ||
+ | </h3></p> | ||
+ | |||
+ | <p><h3>Gel purification</h3></p> | ||
+ | |||
+ | <p>Using Zymoclean Gel DNA Recovery Kit</p> | ||
+ | |||
+ | <p>1. Add 3 volumes (µl) for each volume of agarose (mg) excised from the gel.</p> | ||
+ | |||
+ | <p>2. Incubate at 55°C for 10 minutes until gel is dissolved</p> | ||
+ | |||
+ | <p>3. Pipette into a Zymo-Spin Column & Collection Tube</p> | ||
+ | |||
+ | <p>4. Centrifuge (at 16000 x g)for 1 minute, and discard the flow-through.</p> | ||
+ | |||
+ | <p>5. Add 200µl DNA wash Buffer and centrifuge for 30 seconds.</p> | ||
+ | |||
+ | <p>6. Add another 200µl DNA wash buffer and centrifuge for a minute.</p> | ||
+ | |||
+ | <p>7. Place the spin column into a new sterile microcentrifuge tube. </p> | ||
+ | |||
+ | <p>8. Add up to 30µl of Cloning Water directly to the column matrix.</p> | ||
+ | |||
+ | <p>9. Let it sit for 4 minutes, and centrifuge for 4 minutes.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Treating with DPNI</h3></p> | ||
+ | |||
+ | <p>1. Add to a PCR rxn mix after PCR.</p> | ||
+ | |||
+ | <p>2. 1µl DPNI per 50 µl rxn mix.</p> | ||
+ | |||
+ | <p>3. Let it run 37°C for 1 hour.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>DNA Purification</h3></p> | ||
+ | |||
+ | <p>Using Zymoclean DNA Clean & Concentrator</p> | ||
+ | |||
+ | <p>1. Add 5 volumes of DNA binding Buffer to PCR product or Short DNA fragments.</p> | ||
+ | |||
+ | <p>2. Vortex Tube.</p> | ||
+ | |||
+ | <p>3. Load mixture into Zymo-Spin Column and Collection Tube</p> | ||
+ | |||
+ | <p>4. Centrifuge (16000 x g) for 30 seconds. Discard flow-through.</p> | ||
+ | |||
+ | <p>5. Add 200µl of DNA Wash Buffer and centrifuge for 30 seconds.</p> | ||
+ | |||
+ | <p>6. Add another 200µl of DNA Wash Buffer and centrifuge for 2 minutes.</p> | ||
+ | |||
+ | <p>7. Place the spin column into a new sterile microcentrifuge tube.</p> | ||
+ | |||
+ | <p>8. Add up to 30µl of Cloning Water directly to the column matrix.</p> | ||
+ | |||
+ | <p>9. Let it sit for 4 minutes, and centrifuge for 4 minutes.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Golden Gate Digestion/Ligation</h3></p> | ||
+ | |||
+ | <p>1. Design Primers accordingly, taking into account Type IIs Restriction Enzyme site.</p> | ||
+ | |||
+ | <p>2. Add purified pieces you want to ligate based on concentration results (100ng / x ng/µl) into a PCR tube.</p> | ||
+ | |||
+ | <p>3. Add cloning water to the mix up to 10.5µl total.</p> | ||
+ | |||
+ | <p>4. Add 1.5µl Cutsmart Buffer to the mix.</p> | ||
+ | |||
+ | <p>5. Vortex ligase buffer. Add 1.0µl to the mix.</p> | ||
+ | |||
+ | <p>6. Add 1µl of Type IIs restriction enzyme (we usually use sapI)</p> | ||
+ | |||
+ | <p>7. Add 1µl of T4 Ligase to the mix.</p> | ||
+ | |||
+ | <p>8. Total should be 15µl in the tube. If volume of pieces you want to ligate together add up to greater than 10.5µl, scale up the mix proportionally.</p> | ||
+ | |||
+ | <p>Reaction assembly</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>50 x</p></td> | ||
+ | <td valign="top"> <table border="1" cellspacing="0" cellpadding="0"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td valign="top"><p>37°C</p></td> | ||
+ | <td valign="top"><p>3 min</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>16°C</p></td> | ||
+ | <td valign="top"><p>4 min</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>50°C</p></td> | ||
+ | <td valign="top"><p>5 min</p></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td valign="top"><p>80°C</p></td> | ||
+ | <td valign="top"><p>5 min</p></td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Restriction Enzyme Digestion</h3></p> | ||
+ | |||
+ | <p>1. Follow NEB protocols.</p> | ||
+ | |||
+ | <p>2. Find appropriate buffer if doing double digests.</p> | ||
+ | |||
+ | <p>3. Heat inactivate at the end.</p> | ||
+ | |||
+ | <p>4. If doing sequential digests, heat inactivate in between each step.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Blunt End Ligation</h3></p> | ||
+ | |||
+ | <p>1. Add 18µl of purified PCR product into a PCR tube.</p> | ||
+ | |||
+ | <p>2. Add 2.25µl of 10x T4 Ligase Buffer</p> | ||
+ | |||
+ | <p>3. Add 1.125µl of T4 PolyNucleotide Kinase.</p> | ||
+ | |||
+ | <p>4. Put in thermocycler for 37°C for 45 minutes.</p> | ||
+ | |||
+ | <p>5. Add 1.125µl T4 DNA Ligase</p> | ||
+ | |||
+ | <p>6. Leave at room temperature for at least 1 hour.</p> | ||
+ | |||
+ | <p>7. Heat inactivate whatever you are not using at 65°C for 20 minutes.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Transformation (Electroporation)</h3></p> | ||
+ | |||
+ | <p>1. Put electroporation cuvette on ice and thaw electro-competent cells for 5 minutes.</p> | ||
+ | |||
+ | <p>2. Prepare 500µl LB and a labeled culture tube.</p> | ||
+ | |||
+ | <p>3. Add up to 5µl of DNA directly to the competent cells. (usually use 2µl)</p> | ||
+ | |||
+ | <p>4. Transfer mix into cuvette, ensuring no bubbles and mix touching both sides.</p> | ||
+ | |||
+ | <p>5. Wipe down cuvette to remove moisture.</p> | ||
+ | |||
+ | <p>6. Place in electroporation machine and start.</p> | ||
+ | |||
+ | <p>7. If arc occurs, try with smaller amounts of DNA.</p> | ||
+ | |||
+ | <p>8. If successful, immediately add the LB directly into the cuvette.</p> | ||
+ | |||
+ | <p>9. Transfer LB + transformed cells into the culture tube.</p> | ||
+ | |||
+ | <p>10. Record time and incubate in the 37°C shaker for 1 hour.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Plating</h3></p> | ||
+ | |||
+ | <p>1. Prepare two appropriate antibiotic resistance “100µl” and “Rest” </p> | ||
+ | |||
+ | <p>2. Add 100µl of transformed culture (after the 1 hour incubation) onto the 100µl plate.</p> | ||
+ | |||
+ | <p>3. Use a sterile spreader to streak the liquid culture onto the plate in the order on the left:</p> | ||
+ | |||
+ | <p>4. On the “Rest” Plate, pour the rest of the liquid culture and spread evenly on the plate.</p> | ||
+ | |||
+ | <p>5. Flip and put in the 37°C incubator overnight (no longer than 20 hours).</p> | ||
+ | |||
+ | <p>6. Wrap your plate after sufficiently grown and store in the 4°C fridge for up to 45 days.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Liquid Cultures</h3></p> | ||
+ | |||
+ | <p>1. Determine volume of culture to grow in a culture tube. We usually use 4ml of LB.</p> | ||
+ | |||
+ | <p>2. In the biosafety hood, add 4ml of LB, and 4µl of each appropriate resistance(s).</p> | ||
+ | |||
+ | <p>3. Use a tip to pick up a single colony from the plate.</p> | ||
+ | |||
+ | <p>4. Drop the tip into the media.</p> | ||
+ | |||
+ | <p>5. Cap the culture tube to the first stop.</p> | ||
+ | |||
+ | <p>6. Incubate in 37°C shaker at 250rpm for up to 16 hours.</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Freeze Cultures</h3></p> | ||
+ | |||
+ | <p>1. Add 500µl of 30% glycerol into a cryo-freeze tube.</p> | ||
+ | |||
+ | <p>2. Add 500µl of overnight culture</p> | ||
+ | |||
+ | <p>3. Store in -80°C</p> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p><h3>Plasmid Miniprep</h3></p> | ||
+ | |||
+ | <p>Using Zyppy Plasmid Miniprep Kit </p> | ||
+ | |||
+ | <p>1.Centrifuge overnight culture for 10 minutes at 5000 x g</p> | ||
+ | |||
+ | <p>2. Discard supernatant. Resuspend with 550µl LB</p> | ||
+ | |||
+ | <p>3. Transfer culture to 1.5 ml microcentrifuge tube.</p> | ||
+ | |||
+ | <p>4. Add 100 µl of 7X Lysis Buffer (Blue). Shake well.</p> | ||
+ | |||
+ | <p>5. Within 2 minutes, add 350 µl of cold Neutralization Buffer (Yellow). Shake well.</p> | ||
+ | |||
+ | <p>6. Centrifuge at 16000 x g for 4 minutes.</p> | ||
+ | |||
+ | <p>7. Transfer supernatant into a Zymo-Spin Column and Collection Tube.</p> | ||
+ | |||
+ | <p>8. Centrifuge at 16000 x g for 30 seconds. Discard flow-through.</p> | ||
+ | |||
+ | <p>9. Add 200µl Endo-Wash Buffer. Centrifuge at 16000 x g for 30 seconds.</p> | ||
+ | |||
+ | <p>10. Add 400µl Zyppy Wash Buffer. Centrifuge at 16000 x g for 2 minutes.</p> | ||
+ | |||
+ | <p>11. Transfer column into new labeled microcentrifuge tube.</p> | ||
+ | |||
+ | <p>12. Add 30µl of cloning water directly to column matrix. Let sit for 4 minutes.</p> | ||
+ | |||
+ | <p>13. Centrifuge at 16000 x g for 4 minutes to elute.</p> | ||
</td> | </td> | ||
Line 46: | Line 565: | ||
<table id="general"> | <table id="general"> | ||
<tr> <td> | <tr> <td> | ||
- | <h1> Rebstock Group Protocols </h1> | + | <center> <h1> Rebstock Group Protocols </h1> </center> |
<a name="2"></a> | <a name="2"></a> | ||
Revision as of 03:25, 4 September 2014