Disposable Electroelution System

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Post on 01-Jun-2015

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Demonstrates an extreme low cost solution to prep-scale oligo electroelution that John Picuri and I came up with to save time and improve yield on our DNA prep process.

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<ul><li> 1. The Incredible, Disposable, Electroelution System. All the benefits of electro-elution without the hassle of a setting up and cleaning a full rig and trap. 5/7/07Brian Frezza, John Picuri, and The TSRI Instrumentation and Design Lab</li></ul> <p> 2. Non-disposable Parts Acrylic Sheath Plastic Cap Rubber Cap Rubber O-Ring Acrylic O-Ring 3. Acrylic Sheath 4. Plastic Cap 5. Acrylic O-Ring 6. Rubber O-Ring 7. Rubber Cap 8. Required Equipment Disposable Tea Bag Paper Disposable 20mL Syringe 2 Platinum Electrodes Disposable50mL Beaker Disposable BT1 Membrane 9. Step 1: Cut 20mL syringe at the 13mL mark. Cut here 10. Step 2: Place Acrylic O-Ring on Tea-bag paper and fit into top of syringe. 11. Step 3: Cut away excess Tea-bag Paper and slide Acrylic O-Ring to the bottom of the syringe 12. Step 4: Remove BT1 Membrane from package and punch out a size 8 circle. 13. Step 5: Thread the acrylic sheath into the small hole in the plastic Cap.Then place the rubber O-ring on top of acrylic sheath, and the circular BT1 membrane on top of both. Plastic Cap Acrylic sheath Rubber O-ring Circular BT1 Membrane 14. Step 7: Fit the green rubber cap on top of the circular BT1 membrane such that it seals tightly.Then invert the plastic cap and place on top of disposable 50mL beaker. 15. Step 8: Fill the beaker with ~30mL of TE (Tris, EDTA, pH 7.5) buffer. Then gently fill the acrylic sheath full of TE with a pipette. 16. Step 8: Insert the prepared syringe from step 3 into the large hole in the plastic cap and then pipette the syringe full of TE. 17. Step 9: Place gel slice with DNA in 10mL falcon tube and crush into little pieces with a glass rod. DNA dyed pink (Cy3) for easy visualization 18. Step 10: Invert the tube over the syringe and gently tap to transfer crushed gel. 19. Get psyched, youre almost ready to elute! 20. Step 11: Place positive electrode into the acrylic sheath, and negative electrode into the syringe. 21. Step 12: Check to make sure that both electrodes are submerged and that there are no air bubbles blocking the opening to the syringe or the sheath.Both electrodessubmerged BAD! Air bubble,remove by taping Negative electrode(repels DNA) Positive Electrode (attracts DNA) 22. Step 13: Turn the power on and adjust to 250V.Wait ~5 min per 5mL crushed gelfor complete elution.The BT1 membrane allows salt to pass though it but NOT DNA.So when you turn on the juice, the DNA will be pulled out of the gel into the beaker, but cannot contact the positive electrode since its protected by the sheath and BT1 membrane. 23. Watch the DNA elute! 24. Watch the DNA elute! 25. Watch the DNA elute! 26. Watch the DNA elute! 27. Step 14: Turn off power, remove electrodes and plastic cap.DNA remains in the beaker.No DNA(no color) DNA! 28. Note, there really is no DNA in the sheath or syringeNo DNA(no color) 29. Wheres all the DNA?In the beaker, ready to go! 30. Step 15: Remove and wash, O-rings, sheath, and cap with water and Methanol (optional) 31. Step 16: Toss out all that other junk.Its disposable! 32. Step 17: Marvel at how much time you just saved, and youre greatly improved yields! Remaining DNA in gelafter a48 HourCrush and Soak procedure. Check out all that hard-earned DNA (pink) youre going to have to toss. Remaining DNA in gel after~5 minof electro elution.Can you see any DNA (pink)? 33. Electroelution Comparison : Retail Price: $1,683.55 (starter set, includes whats show from VWR.com) Setup Time: ~20 min Cleanup time: ~30 min The only commercially available electroelution system: Schleicher &amp; SchuellsElutrap Our System: Materials Cost: ~$8.00 Setup time: ~15 min Cleanup time: ~ 5 min 34. Further Optimization </p> <ul><li>Mass production of the syringe prepared in steps 1-3, and the sheath prepared in steps 4-7 would reduce prep time to~3 min .</li></ul> <ul><li>If the acrylic sheath was made of plastic its cost should be low enough (&lt; $1) that all components but the beaker cap could be disposable reducing the cleanup time to~30 seconds. </li></ul> <ul><li>If commercially produced, the electrodes on the cap should be protected by an insulated plug to make it more difficult to shock yourself. </li></ul> <ul><li>All said and done, thesetup cost for the cap w/guides and electrodes would be ~$10 , and a kit containing all other disposable peaces would cost&lt; $1 per run. </li></ul>