Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

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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>
                     <li><a name="prot14" >4CL:STS Sequencing</a></li>
                     <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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- 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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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/>
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 rnxs:<br/><br/>
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For (3) 50 ul rxns:<br/><br/>
- 99.75 ul H2O<br/>
- 99.75 ul H2O<br/>
- 30.00 ul HF Buffer<br/>
- 30.00 ul HF Buffer<br/>
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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 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.