Team:Johns Hopkins/Notebook/Protocols

From 2011.igem.org

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======Vitamin C Concentration Assay======
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======DNA Synthesis======
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(By Anne Marie Helmenstine, Ph.D)
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=====Gel Extraction (QIAquick)=====
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# Excise DNA fragment from agarose gel with clean, sharp scalpel.
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# Weigh gel slice in colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100µl)
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# Incubate @ 50C for 10 min.
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#* SOLUBILIZE AGAROSE COMPLETELY.
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#* To help dissolve gel, vortex tube every 2-3 min during incubation.
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#  After the gel has dissolved, check that the color of the mixture is yellow. If not, add 10µl 3M NaOAc. Or just add the NaOAc anyway.
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# Add 1 gel volume of isopropanol to the sample and mix
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#* Do only if <500bp or >4kb
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# Place spin column in 2mL collection tube.
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# To bind DNA, apply sample to column. Centrifuge 1 min.
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# Discard flowthrough and place column back in same collection tube.
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# Recommended: Add 0.5mL of Buffer QG to column and centrifuge for 1 min.
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# To wash, add 0.75mL of Buffer PE to column and centrifuge for 1 min.
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# Discard flowthrough and centrifuge for an additional 1 min @ 17900 x g (13000rpm).
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# Place column into a clean 1.5mL microcentrifuge tube.
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# To elute DNA, add 30µL Buffer EB to center of membrane, let column stand for 1 min, then centrifuge for 1 min.
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1. Add 25.00 ml of vitamin C standard solution to a 125 ml Erlenmeyer flask.  
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=====Mini-Prep (QIA)=====
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# Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube.
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2. Add 10 drops of 1% starch solution.  
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# Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
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# Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.  
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3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.  
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# Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
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# Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
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4. Titrate the solution until the endpoint is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.  
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# Centrifuge for 30-60s. Discard the flow-through.
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# Recommended: Wash the QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging for 30-60s. Discard flow-through.
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5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.  
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# Wash QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging 30-60s.
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# Discard flow-through and centrifuge for an additional minute to remove residual wash bufffer. **IMPORTANT: Residual wash buffer will not be completly removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzyme reactions.**
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6. Repeat the titration at least twice more. The results should agree within 0.1 ml.  
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# Ordered List ItemPlace QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 µL Buffer EB (10mM Tris-CL, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
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7. You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the endpoint.
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======DNA Assay======
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Revision as of 00:51, 25 September 2011

VitaYeast - Johns Hopkins University, iGEM 2011

Related Links:
Other Protocols:
Vitamin Assays

Contents

DNA Synthesis
Gel Extraction (QIAquick)
  1. Excise DNA fragment from agarose gel with clean, sharp scalpel.
  2. Weigh gel slice in colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100µl)
  3. Incubate @ 50C for 10 min.
    • SOLUBILIZE AGAROSE COMPLETELY.
    • To help dissolve gel, vortex tube every 2-3 min during incubation.
  4. After the gel has dissolved, check that the color of the mixture is yellow. If not, add 10µl 3M NaOAc. Or just add the NaOAc anyway.
  5. Add 1 gel volume of isopropanol to the sample and mix
    • Do only if <500bp or >4kb
  6. Place spin column in 2mL collection tube.
  7. To bind DNA, apply sample to column. Centrifuge 1 min.
  8. Discard flowthrough and place column back in same collection tube.
  9. Recommended: Add 0.5mL of Buffer QG to column and centrifuge for 1 min.
  10. To wash, add 0.75mL of Buffer PE to column and centrifuge for 1 min.
  11. Discard flowthrough and centrifuge for an additional 1 min @ 17900 x g (13000rpm).
  12. Place column into a clean 1.5mL microcentrifuge tube.
  13. To elute DNA, add 30µL Buffer EB to center of membrane, let column stand for 1 min, then centrifuge for 1 min.
Mini-Prep (QIA)
  1. Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube.
  2. Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
  3. Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
  5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
  6. Centrifuge for 30-60s. Discard the flow-through.
  7. Recommended: Wash the QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging for 30-60s. Discard flow-through.
  8. Wash QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging 30-60s.
  9. Discard flow-through and centrifuge for an additional minute to remove residual wash bufffer. **IMPORTANT: Residual wash buffer will not be completly removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzyme reactions.**
  10. Ordered List ItemPlace QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 µL Buffer EB (10mM Tris-CL, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
DNA Assay

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