Team:Calgary/Notebook/Protocols/Process22

From 2011.igem.org

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<li>Add 4 ml of IP-Buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.1% SDS)
<li>Add 4 ml of IP-Buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.1% SDS)
<li>Add PMSF to a final concentration of 1mM
<li>Add PMSF to a final concentration of 1mM
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<li>Sonicate so that DNA fragments are <1 kb on average (9x30sec. at 15% total output appeared to
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<li>Sonicate the solutions 9 times 30 sec. with 30 sec. of rest in between using a Branson 450 sonicator at an output of 15%.
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SONICATION TIMES: ______________ ________________ _________________
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<li>Clarify the solution by centrifugin at 14,000 RPM for 15 min. at 4°C
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12. For the regular IP samples:  Pipette the sonicated solution into 1.5 mL Epindorf tubes and spin them down at 4 degrees (in the cold room if there is a centrifuge there) in a table top centrifuge at 14,000 rpm for 15 min. Once it is finished collect the supernatant and pool it into 15 mL Falcon tubes.  Leave out _______ mL of lysate and freeze the rest immediately in the -80C Freezer.
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<li>Determine protein concentration by Bradford Assay.
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LABELLING HINT! Make sure that you have the Date, Initials, Name of the Lysate and what it exactly is on the tubes. You will have to go back to do this, but also write the protein concentration of the lysate which you will do by Bradford Assay in a few steps. Also parafilm the tops
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For the crosslinked IP samples: Centrifuge at 12,000 g 4C for 10 min.
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FROM THIS STEP ONWARDS IT IS CRITICAL THAT EVERYTHING REMAIN ON ICE AT ALL TIMES. IF NOT THEN YOUR PROTEINS AND DNA WILL GET DEGRADED.
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From this point on the protocols are going to become a little more separated and it may be hard to budget your time effectively.  Hopefully by this point I can come over and help you guys but it should be pretty easy.
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13. Perform a Bradford assay and determine the concentration of the lysates that you have made for regular IP.  Make sure that you write it down.  If you need to know how to do this ask Deirdre.  She should have all the stuff for it.  Don’t worry about doing this for the other lysates (that were crosslinked for chIP) just move on to the next step for them.
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14. Add 1.3 mL ________ mg of regular non-crosslinked lysates (putida and flourescens) to two of the prepped magnetic beads (one which has the biotinylated reaction that you did yesterday, and the other one the control biotin beads – labeling is important here) that you have made and resuspend the beads gently with a pipette.  To the other two tubes add 800 uL of the crosslinked samples to two of the tubes.
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15. Add 8 uL of 100 mM PMSF (stored in the -20 Freezer in the iGEM lab) to the crosslinked tubes and 13 uL of PMSF to the regular IP tubes.  Let them go EOE at 4C O/N in the cold room.  Make sure the tops are well closed.
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Revision as of 05:15, 28 September 2011


Biotinylation and (Theoretical) Immunoprecipitation of Cyclohexanepentanoic Acid

Day 1

1. Inoculate Cultures in 50 mL Tubes (whatever cultures you are interested in probing) with and without the compound you are sensing.

2. At the same time set up a biotinylation reaction as follows, dissolving all solutions in 80% DMSO:

  • Add10 uL of pure cyclohexanepentanoic acid to 167.10 uL of 80% DMSO
  • Make up a solution 500 mM EDAC in 80% DMSO. (Dissolve 10 mg of EDAC in 0.1 mL of 80% DMSO) Add 12.5 uL of the EDAC to the cyclohexanepentanoic acid solution you made. Don’t keep the EDAC out very long it is very hydroscopic and must be kept away from moisture. Store the solution you make at -20C in the biotin box!
  • Make up a stock solution of 100 mM Biotin by weighing it out into a 1.5 mL Epindorf on an analytical balance (be careful when you do this) spin down the tube then add 80% DMSO as before. Again store this solution at -20C in a tightly sealed container.
  • pH the solution using 5% HCl (found in the acid cabinet) to pH 4-7. Optimal pH of the solution appears to be between 5-5.5. Add 2 uL of the acid to the solution and mix well. Take a few microlitres of the solution and spot it onto a piece of pH paper. Repeat if necessary
  • Let the solution sit at room temperature O/N.


3. Set up a second reaction for control with biotin only in 80% DMSO.

  • Add 12.5 uL of the Biotin solution that you made into 187.5 uL of 80% DMSO. pH the solution to roughly the same pH as your reaction using 5% HCl (using ~2uL) and let it sit O/N like the other reaction at room temperature.


FINAL CONCENTRATION OF THE SOLUTION

10 mg of cyclohexane pentanoic acid (or 54mM) 5 mM EDAC

5 mM Biotin

Day 2

Continue the biotinylation from the previous day:
4. After the 24 hours, take 25 uL of the solutions and freeze them for HPLC analysis.
5. Take both biotinylation solutions and dilute the samples in 1.8 mL of PBS solution (50 mM Sodium Phosphate, 15 mM NaCl, pH 8.2), mix well.
6. Add 50 uL of pre-washed streptavidin coated magnetic beads (SoluLink) to each solution and allow them to go end-over-end for 2 hours at room temperature.
7. After incubation, apply the magnet to the side of the tube and wait for the beads to collect (this should only take about 1 min. but make sure you get them all). Remove the solution using a pipet tip being very careful not to touch the beads. Wash the beads with 1 mL of PBS.
Prepare Lysates of Your Favorite Organism: (Following is the lysate protocol we used for producing Pseudomonas lysates adapted from WikiProtocols href="http://www.openwetware.org/wiki/ChIP-Chip_E._coli).

  • Once your cells have grown and reached an optical density OD600 of at least 0.5 (O/N should have been good enough but you might want to check just in case), centrifuge 2500xg, 4°C, 10 min.
  • For each tube, wash twice in cold 10 ml TBS pH 7.5 (20mM Tris, 150 mM NaCl) (You can freeze the cell pellet and proceed later if required).
  • Resuspend in 1 ml Lysis-Buffer (10mM Tris pH 8.0, 20% sucrose, 50mM NaCl, 10mM EDTA, 10 mg/mL lysozyme)
  • Incubate at 30 °C for 30 min (not shaking)
  • Add 4 ml of IP-Buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.1% SDS)
  • Add PMSF to a final concentration of 1mM
  • Sonicate the solutions 9 times 30 sec. with 30 sec. of rest in between using a Branson 450 sonicator at an output of 15%.
  • Clarify the solution by centrifugin at 14,000 RPM for 15 min. at 4°C
  • Determine protein concentration by Bradford Assay.

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