SOLiD™ EZ Bead™ Emulsifier

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File:Solid_EZ_Bead_System_Emulsifier.png|500px

[править] Choose the scale of preparation

You can prepare templated beads according to the amount of library that you want to amplify:

  

Scale of preparationAverage yield of templated beads
E10125 million
E20250 million
E801 billion


[править] Materials and equipment required

[править] Required kits (E10)

  Required Applied Biosystem kits (E10):

Item (part number) Components
SOLiD™ EZ Bead™ Emulsifier E10 Reagent Kit (4452720)Box 1:
•SOLiD™ EZ Bead™ Emulsifier E10 - Aqueous Master Mix
•SOLiD™ EZ Bead™ Emulsifier E10 - P1 Beads

Box 2:

•SOLiD™ EZ Bead™ Emulsifier E10 - P1 reagent
•SOLiD™ EZ Bead™ Emulsifier E10 - P2 reagent
•SOLiD™ EZ Bead™ Emulsifier E10 - BB Solution

Box3:

•SOLiD™ EZ Bead™ Emulsifier Reagent - 1xTEX Buffer
•SOLiD™ EZ Bead™ Emulsifier Reagent - 1x Low TE Buffer
SOLiD™ EZ Bead™ Emulsifier Oil Pack Kit (4457185)SOLiD™ EZ Bead™ Emulsifier Oil Kit:
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 1
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 2
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil

SOLiD™ EZ Bead™ Emulsifier Oil Accessory Kit:

•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Mix Bottle
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Funnel
SOLiD™ EZ Bead™ Emulsifier E10 Accessories Kit (4453065)
•SOLiD™ EZ Bead™ Emulsifier E-Small Jar Pack
•SOLiD™ EZ Bead™ Emulsifier Oil Kit E-Small AQ Tube Pack
•SOLiD™ EZ Bead™ Emulsifier Tubing Pack
SOLiD™ EZ Bead™ E10 System Consumables, 4-pack (4453093)*
•SOLiD™ EZ Bead™ Emulsifier E10 reagent Kit
•SOLiD™ EZ Bead™ Emulsifier E10 Accessories Kit
•SOLiD™ EZ Bead™ Enricher Buffer Kit

 *The kit includes consumables for all three instruments of the SOLiD™ EZ Bead™ System:the SOLiD™ EZ Bead™ Emulsifier, SOLiD™ EZ Bead™ Amplifier and the SOLiD™ EZ Bead™ Enricher


[править] Required kits (E20)

  Required Applied Biosystem kits (E20):

Item (part number) Components
SOLiD™ EZ Bead™ Emulsifier E20 Reagent Kit (4452721)Box 1:
•SOLiD™ EZ Bead™ Emulsifier E20 - Aqueous Master Mix
•SOLiD™ EZ Bead™ Emulsifier E20 - P1 Beads

Box 2:

•SOLiD™ EZ Bead™ Emulsifier E20 - P1 reagent
•SOLiD™ EZ Bead™ Emulsifier E20 - P2 reagent
•SOLiD™ EZ Bead™ Emulsifier E20 - BB Solution

Box3:

•SOLiD™ EZ Bead™ Emulsifier Reagent - 1xTEX Buffer
•SOLiD™ EZ Bead™ Emulsifier Reagent - 1x Low TE Buffer
SOLiD™ EZ Bead™ Emulsifier Oil Pack Kit (4457185)SOLiD™ EZ Bead™ Emulsifier Oil Kit:
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 1
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 2
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil

SOLiD™ EZ Bead™ Emulsifier Oil Accessory Kit:

•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Mix Bottle
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Funnel
SOLiD™ EZ Bead™ Emulsifier E20 Accessories Kit (4453076)
•SOLiD™ EZ Bead™ Emulsifier E-Small Jar Pack
•SOLiD™ EZ Bead™ Emulsifier Oil Kit E-Small AQ Tube Pack
•SOLiD™ EZ Bead™ Emulsifier Tubing Pack
SOLiD™ EZ Bead™ E20 System Consumables, 4-pack (4453094)*
•SOLiD™ EZ Bead™ Emulsifier E20 reagent Kit
•SOLiD™ EZ Bead™ Emulsifier E20 Accessories Kit
•SOLiD™ EZ Bead™ Enricher Buffer Kit

 *The kit includes consumables for all three instruments of the SOLiD™ EZ Bead™ System:the SOLiD™ EZ Bead™ Emulsifier, SOLiD™ EZ Bead™ Amplifier and the SOLiD™ EZ Bead™ Enricher


[править] Required kits (E80)

  Required Applied Biosystem kits (E80):

Item (part number) Components
SOLiD™ EZ Bead™ Emulsifier E80 Reagent Kit (4452722)Box 1:
•SOLiD™ EZ Bead™ Emulsifier E80 - Aqueous Master Mix
•SOLiD™ EZ Bead™ Emulsifier E80 - P1 Beads

Box 2:

•SOLiD™ EZ Bead™ Emulsifier E80 - P1 reagent
•SOLiD™ EZ Bead™ Emulsifier E80 - P2 reagent
•SOLiD™ EZ Bead™ Emulsifier E80 - BB Solution

Box3:

•SOLiD™ EZ Bead™ Emulsifier Reagent - 1xTEX Buffer
•SOLiD™ EZ Bead™ Emulsifier Reagent - 1x Low TE Buffer
SOLiD™ EZ Bead™ Emulsifier Oil Pack Kit (4457185)SOLiD™ EZ Bead™ Emulsifier Oil Kit:
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 1
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Stabilizer 2
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil

SOLiD™ EZ Bead™ Emulsifier Oil Accessory Kit:

•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Mix Bottle
•SOLiD™ EZ Bead™ Emulsifier Oil Kit - Oil Funnel
SOLiD™ EZ Bead™ Emulsifier E80 Accessories Kit (4453070)
•SOLiD™ EZ Bead™ Emulsifier E-Small Jar Pack
•SOLiD™ EZ Bead™ Emulsifier Oil Kit E-Small AQ Tube Pack
•SOLiD™ EZ Bead™ Emulsifier Tubing Pack
SOLiD™ EZ Bead™ E80 System Consumables, 4-pack (4453095)
•SOLiD™ EZ Bead™ Emulsifier E80 reagent Kit
•SOLiD™ EZ Bead™ Emulsifier E80 Accessories Kit
•SOLiD™ EZ Bead™ Enricher Buffer Kit

 *The kit includes consumables for all three instruments of the SOLiD™ EZ Bead™ System:the SOLiD™ EZ Bead™ Emulsifier, SOLiD™ EZ Bead™ Amplifier and the SOLiD™ EZ Bead™ Enricher


[править] Required Material

50ml Conical tubes (Falcon), Ambion #AM12502
1000µl Barrier (filter) tips, Ambion #AM12665
1ml Syringe, Terumo #BS-01T
3ml Syringe, BD

[править] Before you begin

• Construct the DNA library of interest
• Prepare and quantitate the DNA library before proceeding with emulsion preparation. The recommended ePCR library concentrations are 0.1, 0.3 and 0.5pM as starting points.
• Install the SOLiD™ EZ Bead™ Emulsifier in a different location than the location used to enrich the emulsion. Emulsification and enrichment in different locations prevents contamination of the sample.

[править] Prepare and run the SOLiD™ EZ Bead™ Emulsifier

NB! Preparation guide for SOLiD™ EZ Bead™ Emulsifier (E80)

• Items: 50ml Falcon, 1ml syringe, 3ml syringe, SOLiD™ EZ Bead™ Emulsifier Oil Pack Kit
• NB! A single Oil Pack Kit will yield approximately 240ml of Oil Master Mix, which is 6x 50ml Falcon tubes worth

  1.   Preparing SOLiD™ EZ Bead™ Oil Master Mix with vortexer
    » prepare the Oil Master Mix in 6x 50ml Falcon tubes (2samples)/ 3x 50ml Falcons tubes (1sample):
    ItemVolume
    Stabilizer 1 1,8ml
    Stabilizer 2 400µl
    Mineral Oil37,8ml
    Total Volume40ml

    Use a 3ml syringe to dispense 1,8ml of Stabilizer 1 into the 50ml Falcon tube. To avoid air bubbles, aspirate and dispense some reagent before aspirating the correct volume.
    Use a 1ml syringe to dispense 400µl of Stabilizer 2 into the 50ml Falcon tube. Ensure no air bubbles are in the syringe.
    Poor mineral oil into the tube
    » Cap the Falcon tubes and vortex the solution so a whirlpool down to the bottom of the tube is created. Once the whirlpool is created, continue to whirlpool-vortex for another 30-60 seconds
    » Pour the mixtures into the Oil Mix Bottle, you should have approximately 240ml Oil Master Mix
    » Shake the bottle by hand for 30 seconds.
    » If the Oil Master Mix will be used the same day: degas at room temperature for at least 30 minutes in a dark location; if the Oil Master Mix will not be used the same day: degas and store the Oil Master Mix at 4°C, let it come to room temperature for 30 minutes

    You can thaw the SOLiD™ EZ Bead™ Emulsifier reagents on ice during preparation of P1 Beads and install the oil phase

  2.   Prepare the P1 Beads:
    » Vortex and pulse-spin one tube of SOLiD™ EZ Bead™ Emulsifier E80-P1 Beads
    » Ensure that the P1 Beads are completely resuspend, then pipet 1430µl (2x715µl) of P1 Beads into a 2.0ml LoBind tube
    » Place the tube in a magnetic rack for at least 1 minute. After the solutionclears, remove and discard the supernatant.
    » Resuspend the beads in 1430µl SOLiD™ EZ Bead™ Emulsifier E80-BB Solution.
    » Sonicate the beads using the Bead Block Declump program on the Covaris S2 System, the pulse spin the tube
    » Place the tube of beads in a magnetic rack for at least 1 minute. After the solutionclears, remove and discard the supernatant.
    » Resuspend the beads in 1430µl of 1x TEX Buffer.
    » Store the beads at 4°C until ready for use, not on ice
  3.   Install the oil phase
    » Unscrew and retain the cap with impeller of the SOLiD™ EZ Bead™ Emulsifier E-Large Jar
    » Tare the E-Large Jar on an analytical balance
    » Pour 67,9g of Oil Master Mix into the E-Large Jar
    » Screw the cap with the impeller on the jar
    » Install the jar on the SOLiD™ EZ Bead™ Emulsifier:
    a. Turn the knob clockwise to open the compartment door.
    b. Pull the arm back at the base towards the opening of the compartment.
    c. Slide the jar until it stops against the two posts at the rear of the compartment.
    d. Allow the arm to rest against the jar (see Fig.1)
    e. Rotate the jar so that the port on the cap is to the left of the impeller shaft.
    » Fit the impeller head into the locking slot. Slide the coupler down until the coupler clicks to a stop to ensure that the impeller head is firmly in place in the coupler (see Fig.2)
    » Attach the tubing with the Luer-Lok to the port of the cap
    » Insert the 3-stop tubing with one stop in the pump cassette (see Fig.3)
    a. Repeat the step to insert the tubing with the second stop at the other end of the pump cassette. Ensure that the tubing is in the inner groove opposite the second stop (see Fig. 3) and that the tubing is in the correct orientation
    b. Install the pump cassette into the roller-head of the pump.
    c. Turn the pinch wheel in the pump cassette counter-clockwise so that the mark is in the blue zone. This adjustment maintains correct tubing tension for normal pump operation.
    » Position the tubing so that it threads through the tubing tab
    » Close the door of the compartment, then turn the knob counter-clockwise to lock the door.
    File:Emulsifier-jar.png|400px|thumb|left|Fig.1- The jar rests on the back of the groove and the arm is against the jar File:Emulsifier-impeller_head.png|450px|thumb|Fig.2- How to fit the impeller head into the locking slot]]
    File:Emulsifier_tubing.png|500px|thumb|left|Fig.3- How to insert the tubing into the pump cassette]]
  4.   Prepare the aqueous phase
    » Thaw on ice:
    a. SOLiD™ EZ Bead™ Emulsifier E80-Aqueous Master Mix. Do not vortex
    b. SOLiD™ EZ Bead™ Emulsifier E80-P1 Reagent, then vortex the reagent for a few seconds, and pulse-spin
    c. SOLiD™ EZ Bead™ Emulsifier E80-P2 Reagent, then vortex the reagent for a few seconds, and pulse-spin
    » SOLiD™ EZ Bead™ Emulsifier E-Large AQ Tube on ice for filling
    » Prepare a 1:10 dilution of P1 Reagent by adding 21µl of P1 reagent to 189µl of Low TE Buffer in a 1.5ml LoBind Tube
    » Add the appropiate volume to the AQ Tube in this order:
    E80 ReagentVolume (µl)
    Aqueous Master Mix 4800-x
    1:10 diluted P1 Reagent200
    Undiluted P2 Reagent300
    Library templatex
     *x= Volume of library template.
    » Cap the AQ Tube and swirl to mix the solution. Place the AQ Tube on ice
  5.   Set up and run the SOLiD™ EZ Bead™ Emulsifier
    » Turn on the SOLiD™ EZ Bead™ Emulsifier
    » Ensure that the Oil Master Mix in the SOLiD™ EZ Bead™ Emulsifier Jar is at room temperature
    » Vortex, then pulse-spin the tube of prepared P1 beads
    » Sonicate the prepared P1 beads using the Covalent Declump 3 program on the Covaris S2 System, then pulse-spin the tube.
    Perform the next steps as quickly as possible, before the beads settle in the aqueous phase! If the beads settle, swirl the aqueous phase again
    » Immediately add the beads to the aqueous phase, then swirl the AQ Tube until the suspension is uniformly brown
    » Immediately load the AQ Tube onto the SOLiD™ EZ Bead™ Emulsifier, attach the tubing to the cap at the AQ Tube. Ensure that the beads have not settled.
    » Start the run:
    a. Press the scale-selection button to select E80 (see Fig.4)
    b. Press the start button. The motor starts and runs for 10 seconds before the pump starts. The middle light on the panel is lit yellow.
    Do not open the door of the emulsifier while a run is in progress. Opening the door automatically aborts the run and the emulsion is not properly prepared.

    The run time is approximately 15 minutes. After the run is complete the SOLiD™ EZ Bead™ Emulsifier stops automatically, the right light on the panel is lit green, and the panel says "Complete".
    File:Emulsifier_key_panel.png|500px|thumb|left|Fig.4- Emulsifier key panel]]
  6.   Collect the emulsion and perform an emulsion PCR
    » Ensure that the run is finished. When the run is finished, the clock says "00:00."
    » Turn the knob clockwise to unlock the compartment door, then open the door.
    » Slide the coupler up and away from the impeller head.
    » Gently pull the impeller head from the cuopler.
    » Swing out the arm at the base, then remove the impeller and the jar fromthe compartment.
    » Detach the tubing from the port on the cap of the jar.
    » Remove the pump cassete, tubing and AQ Tube from the instrument as one unit.
    » To release the tubing from the pump cassette, turn the pinch wheel clockwise to the silver zone, then remove the tubing.
    » Discard the used AQ Tube and tubing. Store the pump cassette on the top of the SOLiD™ EZ Bead™ Emulsifier.
    The pump cassette is part of the instrument so is not disposable.
    » Close the door of the compartment, then turn the knob-counter clockwise to close the door.
    » Immediately perform emulsion PCR (ePCR) on the emulsion using the SOLiD™ EZ Bead™ Amplifier


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