Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

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     <div id="protocol-container">
     <div id="protocol-container">
             <div id="protocolselection">
             <div id="protocolselection">
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<center><h2>Protocol Selection</h2></center>
                     <li><a name="prot1" >Bootcamp Protocols</a></li>
                     <li><a name="prot1" >Bootcamp Protocols</a></li>
                     <li><a name="prot2" >Digestions</a></li>
                     <li><a name="prot2" >Digestions</a></li>
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                     <li><a name="prot12" >Subculturing Plates</a></li>
                     <li><a name="prot12" >Subculturing Plates</a></li>
                     <li><a name="prot13" >Transformation of E.Coli</a></li>
                     <li><a name="prot13" >Transformation of E.Coli</a></li>
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                    <li><a name="prot16" >mCherry Experiments</a></li>
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                    <li><a name="prot14" >4CL:STS Sequencing</a></li>
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                    <li><a name="prot15" >Biosynthesis of piceatannol</a></li>
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             </div>
                  
                  
             <div id="protocoloverview">
             <div id="protocoloverview">
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<h2>Making LB for plates</h2> <br/>
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<center><h2>UIUC iGEM Protocols</h2></center>
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To make 1 Liter of LB: <br/><br/>
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1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
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2. Add dry ingredients first. <br/>
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3. Use a 2L flask. <br/> <br/>
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4. Add the following <br/><br/>
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- 10 g Tryptone <br/>
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- 5 g Yeast Extract <br/>
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- 5 g NaCl <br/>
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- 1.5 g Agar (NOT agarose!)<br/><br/>
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4. Then add 1 L of MilliQ water. <br/>
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5. Autoclave by total volume.<br/>
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6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
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<h2>Making Liquid Media </h2> <br/>
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<center><p>The standard protocols for each technique used in our project endeavors have been documented. Unless further noted <a href="http://www.scs.illinois.edu/rao/index.php">all procedures are based off of those used by the lab of C. V. Rao</a>.</p><br/></center>
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1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/>
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2. Add dry ingredients first, then add MilliQ water <br/>
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3. No Agar! <br/>
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4. Autoclave by total volume <br/><br/>
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<h2>Making Glycerol Stock </h2> <br/>
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To make 400 mL of 10% glycerol you will need:<br/><br/>
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-40 mL glycerol<br/>
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-360 mL of MilliQ water <br/><br/>
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To make 400 mL of 20% glycerol you will need:<br/><br/>
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-80 mL glycerol<br/>
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-320 mL of MilliQ water <br/><br/>
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1. Use flasks and bottles before graduated cylinders (they take forever to mix!)<br/>
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2. Need a stir plate and a large stir bar <br/>
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3. Stir until mixture is homogeneous <br/>
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4. Don’t autoclave! <br/><br/>
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<h2>Making ddH2O</h2> <br/>
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- Use the small bottles. <br/>
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- Autoclave water by total volume. <br/><br/>
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<h2>Autoclaving </h2><br/>
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- Robert is the source of official help on all things about the Autoclave. <br/><br/>
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1. Use the autoclave tape. <br/>
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2. Look at the Betastar for settings. <br/>
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3. Go by materials and total volume. <br/>
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4. Make sure the autoclave tape has changed colour by the end of the cycle. Check conditions and settings then repeat steps 1-4 if tape has not changed colour. <br/><br/>
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<h2>Ligation </h2><br/>
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We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/>
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1. Control: <br/><br/>
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- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/>
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- 2 uL T4 ligase buffer<br/>
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- 1 uL T4 ligase <br/><br/>
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2. P&C: <br/><br/>
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- 2 uL of Circular PSB1C3 plasmid<br/>
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- 2 uL of PUF, 11 uL ddH2O<br/>
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- 2 uL T4 ligase buffer<br/>
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- 1 uL T4 ligase<br/><br/>
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3. L&P:<br/><br/>
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- 2 uL of linear PSB1C3 plasmid<br/>
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- 2 uL of PUF<br/>
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- 11 uL ddH2O<br/>
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- 2 uL T4 ligase buffer<br/>
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- 1 uL T4 ligase<br/><br/>
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<div id="prot1" style="display:none">
<div id="prot1" style="display:none">
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- 1 µl – NEB Enzyme 2 (10 Units/µl) = 10 Units in 50 µl reaction<br/>
- 1 µl – NEB Enzyme 2 (10 Units/µl) = 10 Units in 50 µl reaction<br/>
- dH2O to 50 µl<br/>
- dH2O to 50 µl<br/>
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- BSA<br/>
- TOTAL – 50 µl<br/><br/>
- TOTAL – 50 µl<br/><br/>
Procedure:<br/><br/>
Procedure:<br/><br/>
1. Calculate how much template is needed for digestion. Then calculate how much water needs to be added to make a 50 µl reaction.<br/>
1. Calculate how much template is needed for digestion. Then calculate how much water needs to be added to make a 50 µl reaction.<br/>
2. Pipette appropriate amounts of dH20, template, and 10x Buffer in that order into a PCR tube (200 µl). Mix reagents together by vortexing and then tapping tube on desk to keep reagents on the bottom of the PCR tube.<br/>
2. Pipette appropriate amounts of dH20, template, and 10x Buffer in that order into a PCR tube (200 µl). Mix reagents together by vortexing and then tapping tube on desk to keep reagents on the bottom of the PCR tube.<br/>
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3. Add appropriate amounts of Enzyme 1 and Enzyme 2. Once added gently swirl around reaction mix with pipette tip.<br/>
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3. Add appropriate amounts of Enzyme 1 and Enzyme 2. Add .5ul BSA, or other volume depending on BSA content in enzymes. Once added gently swirl around reaction mix with pipette tip.<br/>
4. Incubate at 37o C for 1 – 2 hours<br/>
4. Incubate at 37o C for 1 – 2 hours<br/>
5. Incubate at 80 oC for 20 minutes to deactivate the restriction enzymes <br/>
5. Incubate at 80 oC for 20 minutes to deactivate the restriction enzymes <br/>
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13. Discard the column <br/>
13. Discard the column <br/>
14. Label the collected liquid and store it in the -20˚C freezer until use<br/><br/>
14. Label the collected liquid and store it in the -20˚C freezer until use<br/><br/>
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<h2>Colony PCR</h2><br/>
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What you need:<br/><br/>
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- Autoclaved 1.5 mL centrifuge tubes<br/>
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- Autoclaved MilliQ water<br/>
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- Green master mix<br/>
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- Primers complementary to the gene of interest, that specifically reveal both its presence and its location<br/>
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- PCR strip tubes<br/>
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- An agar plate containing colonies to be tested<br/>
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- Autoclaved toothpicks<br/><br/>
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Procedure:<br/><br/>
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1. In a 1.5mL centrifuge tube, mix 12.5uL*(# of samples) of autoclaved water, 12.5uL*(# of samples) of green mix, and 1uL*(# of samples of each primer<br/>
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2. Add 27uL of the mixture to each tube of the PCR strip tubes<br/>
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3. Number the colonies you wish to test as well as the tubes of liquid<br/>
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4. With an autoclaved toothpick, gently poke each colony and stir it into the corresponding liquid <br/>
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5. Be sure all liquid is in the bottom of the tubes (spin tubes in centrifuge if it isn’t)<br/>
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6. Run the samples in the PCR machine, using the colony PCR program with an extension time of roughly one minute per kb of DNA to be amplified by your primers<br/>
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7. Analyze samples on a gel<br/><br/>
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2. If no colonies are formed plate out the rest of the transformants (~800 µl) and incubate at 37o C for 24 hrs<br/><br/>
2. If no colonies are formed plate out the rest of the transformants (~800 µl) and incubate at 37o C for 24 hrs<br/><br/>
</div>
</div>
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<div id="prot14" style="display:none">
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<center><h2>To sequence the 4CL:STS part from the parts registry</h2></center><br/>
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1. PCR the 4CL:STS mini prep DNA in the plastic box labeled “Other” in the -20C (this will be your template in the PCR rxn) Please do 6 total rxns for a total of 300 ul.<br/><br/>
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For (3) 50 ul rxns:<br/><br/>
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- 99.75 ul H2O<br/>
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- 30.00 ul HF Buffer<br/>
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- 3.00 ul dNTPs<br/>
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- 7.50 ul primer 1 (VF2 is the name 20 bp, TempMelt= 63.8C GC%= 50)<br/>
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- 7.50 ul primer 2 (VF is the name, 20 bp, TempMelt= 63.4C, GC%= 50)<br/>
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- 1.50 ul of template (microfuge tube of 4CL:STS mini prep in the “Other” box in -20C)<br/>
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- 0.75 ul of Phusion (preferably Phusion Hot start)<br/>
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- 150 ul total<br/><br/>
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2. Divide this 150 ul among 3 small PCR tubes<br/><br/>
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Put them in the PCR machine with the conditions:<br/><br/>
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1. 98 deg C for 3:00 min<br/>
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2. 98 deg C for 0:15 sec<br/>
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        3. 64 deg C for 0:15 sec<br/>
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        4. 72 deg C for 1:30 min<br/>
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        * Go back to step 2, repeat 34 times*<br/>
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        5. 72 deg C for 5:00 min<br/>
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        6. 12 deg C for infinty <br/><br/>
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3. Make a 1% Agarose gel to gel purify the PCR, use the small comb that creates large wells, use the 1kb ladder (1.5 ul) in one lane. In another lane use all of the PCR product + 15 ul of 6X loading dye to dye the product. MAKE SURE IT IS A THICK GEL (use approx 50-70 ml of gel)<br/><br/>
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Band calculations:<br/><br/>
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- VF2 amplifies from approx 2020 bp of pSB1A3 to 2155 of pSB1A3= 135 bp<br/>
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- VF amplifies from approx 160 bp of pSB1A3 to 0 bp of pSB1A3= 160 bp<br/>
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- 4CL:STS part is 1815 bp long<br/>
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- TOTAL PCR band product: 2110 bp *look for this size band*<br/><br/>
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4. If you get a 2110 bp band, gel purify this band by cutting out the band and following gel purification protocol.
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if the band looks bright, elute with 50ul of H2O and incubate for 1:00 min<br/><br/>
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- If the band looks faint, elute with 30 ul of H2O and incubate for 5:00 min<br/>
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- If you can barely see the band, elute with 15 ul of H2O and incubate for 5:00 min<br/><br/>
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5. Make a second 1% Agarose gel to test the purification product, use the small comb that creates small wells, use the 1kb ladder (1.5 ul) in one lane. In another lane use 1 ul of the purification product + 1 ul of 6X loading dye to dye the product (this step is to make sure you didn’t lose your product in the purification step)<br/><br/>
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- Now these are the details for the next step. You don’t have to rush on this because hopefully I’ll be back to help you start this https://unicorn.biotec.illinois.edu/Sequencing_Protocol.pdf<br/><br/>
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This is a protocol for the sequencing PCR. We’ll need 75-100ng of DNA in order to start this step, so please do maybe 6 PCRs in step 1 to get enough DNA for this.<br/><br/>
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</div>
</div>
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<div id="prot15" style="display:none">
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<center><h2>Biosynthesis of Piceatannol from Resveratrol in E. Coli</h2></center><br/>
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1. Grow BM3 wt, BM3 10, BM3 13, in 4ml of LB + Amp media overnight from gylcerol stock in -80 C freezer<br/>
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2. Take 250ul of this culture and a dd to 25ml of new LB + 25 ul Amp; grow at 37 C for approx. 2 hours.<br/>
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3. When OD@600nm = 0.4, add isopropyl-Dthiogalactopyranoside (IPTG) to a final concentration of 0.50 mM. For our 25mL samples we added 12.5 ul of IPTG 1M.<br/>
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4. Get to mid log phase OD@600nm > 0.8, this should take approx. 2 hours.<br/>
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5. Transfer contents to Falcon tubes.<br/>
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6. Pellet for 5:00min at 13.2k rpm and resuspend cells in 25ml of this media:<br/><br/>
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- 500ml 1% gylcerol Tris buffer with salene<br/>
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-Tris Buffred Saline (TBS), pH 7.4<br/> of 10 mM Tris and 150 mM NaCl.<br/>
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- 500 ml of TBS can be prepared by dissolving 0.605 g of Tris base and 4.35 g of NaCl in 500 ml of distilled water. Adjust the pH before use.<br/>
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- Add 5ml of glycerol<br/><br/>
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6. Autoclave in 1L container<br/>
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7. Add 0.75 mg resveratrol (solubility 0.03 g/L) meaning a concentration of 0.03 g/L<br/>
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8. Grow at 30 C for 36 h, taking 50ul samples of culture<br/>
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9. Dip the capillary tubes in the 50ul sample and spot on TLC plate<br/>
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10. Put plate in developing chamber, add ~10ml of Normal buffer to the solution. Cover with the aluminum foil. Develop to ~1cm from the top.<br/><br/>
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</div>
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<div id="prot16" style="display:none">
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<center><h2>mCherry Experimental Protocols</h2></center><br/><br/>
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1) Inoculate 2 of each of the following cultures in 5ml LB: <br/><br/>
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BL21 (no antibiotic)<br/><br/>
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A. <br/><br/>
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            mcherry + Wild Type Puf Binding Site in Protet plasmid & Wild Type Puf+Pin <br/>
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            (5ul amp* + 5ul cm*)<br/>
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            mcherry + Wild Type Puf Binding Site in Protet plasmid & Mutant Puf+Pin <br/>
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            (5ul amp + 5ul cm)<br/><br/>
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          B.<br/><br/>
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            mcherry + Mutant Puf Binding Site in Protet plasmid & Wild Type Puf+Pin <br/>
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            (5ul amp + 5ul cm)<br/>
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mcherry + Mutant Puf Binding Site in Protet plasmid & Mutant Puf+Pin <br/>
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            (5ul amp + 5ul cm)<br/><br/>
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          C.<br/><br/>
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mcherry + Control Binding Site in Protet plasmid & Wild Type Puf+Pin <br/>
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            (5ul amp + 5ul cm) <br/>
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            mcherry + Control Binding Site in Protet plasmid & Mutant Puf+Pin <br/>
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            (5ul amp + 5ul cm)<br/>
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<br/>
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Grow cultures for 14-16hrs at 37 degrees C<br/><br/>
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Note: <br/><br/>
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*amp: Ampicillin 100 <br/>
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*cm: Chloramphenicol 34<br/>
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<br/>
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2) Subculture each culture by adding 50ul of overnight culture into 5ml LB (with appropriate antibiotics as step one). Since there are two of each culture, label one of each duplicate “induced” and the other duplicate “un-induced”.
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<br/><br/>
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Grow cultures for 3 hrs at 37 degrees C<br/>
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<br/>
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3) Induce the cultures labeled “induced” by adding 1.5ul of the IPTG stock (stock is 1M) for a final concentration of 300 uM IPTG.
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<br/><br/>
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Grow cultures for 6 hrs at 37 degrees C<br/>
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<br/>
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4) Use the Fluorescence plate reader<br/><br/>
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Latest revision as of 06:30, 26 October 2012

Header

Protocols

Protocol Selection

  • Bootcamp Protocols
  • Digestions
  • Gel Purification
  • Inoculation
  • Ligation
  • Making Electrocompetent E.Coli
  • Making Electrophoresis Gels
  • Making TAE Buffers
  • Miniprep
  • PCR Protocols
  • Storage of Cells
  • Subculturing Plates
  • Transformation of E.Coli
  • mCherry Experiments
  • 4CL:STS Sequencing
  • Biosynthesis of piceatannol
  • UIUC iGEM Protocols

    The standard protocols for each technique used in our project endeavors have been documented. Unless further noted all procedures are based off of those used by the lab of C. V. Rao.