SOLiD™ EZ Bead™ Enricher

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[править] Materials and equipment required

[править] Required kits (E10)

  Required Applied Biosystem kits (E10):

Item (part number) Components
SOLiD™ EZ Bead™ Enricher E10 Reagent Kit (4452723)Box 1:
•SOLiD™ EZ Bead™ Enricher E10 – Capture Beads

Box 2:

•SOLiD™ EZ Bead™ Enricher E10 – Capture Oligo
SOLiD™ EZ Bead™ Enricher Buffer Kit (4444140)Box 1:
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 2
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 3
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Rinse Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Hyb Buffer

Box 2:

• SOLiD™ EZ Bead™ Enricher Buffer Kit – Capture Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Release Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1

Additive
Box 3:

•SOLiD™ EZ Bead™ Enricher Buffer Kit – Butanol§
SOLiD™ EZ Bead™ Enricher Accessories Kit (4453073)
• SOLiD™ EZ Bead™ Enricher Column
• SOLiD™ EZ Bead™ Enricher Input Tube
• SOLiD™ EZ Bead™ Enricher Bead Output Tube
• SOLiD™ EZ Bead™ Enricher QC Output Tube
• SOLiD™ EZ Bead™ Enricher Hyb Tube
• SOLiD™ EZ Bead™ Enricher Capture Oligo Tube
• SOLiD™ EZ Bead™ Enricher Release Tube
• SOLiD™ EZ Bead™ Enricher Capture Bead Tube
• SOLiD™ EZ Bead™ Enricher Unenriched Bead Bottle
• SOLiD™ EZ Bead™ Enricher Filter
• SOLiD™ EZ Bead™ Sipping Assembly
SOLiD™ Pre Deposition Kit (4452805)
•10✕ Terminal Transferase Buffer
•10✕ Cobalt Chloride
•Terminal Transferase
•Bead Linker
SOLiD™ EZ Bead™ E10 System Consumables, 4-pack (4453093)*
• SOLiD™ EZ Bead™SOLiD EZ Bead Emulsifier E10 Accessories Kit
• SOLiD™ EZ Bead™ Amplifier E10 Accessories Kit
• SOLiD™ EZ Bead™ Enricher E10 Reagent Kit
• SOLiD™ EZ Bead™ Enricher Buffer Kit
• SOLiD™ EZ Bead™ Enricher Accessories Kit

 § Kit contains 2-butanol.
 *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™ Enricher E20 Reagent Kit (4452724)Box 1:
•SOLiD™ EZ Bead™ Enricher E20 – Capture Beads

Box 2:

•SOLiD™ EZ Bead™ Enricher E20 – Capture Oligo
SOLiD™ EZ Bead™ Enricher Buffer Kit (4444140)Box 1:
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 2
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 3
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Rinse Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Hyb Buffer

Box 2:

• SOLiD™ EZ Bead™ Enricher Buffer Kit – Capture Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – Release Buffer
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1
• SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1

Additive
Box 3:

•SOLiD™ EZ Bead™ Enricher Buffer Kit – Butanol§
SOLiD™ EZ Bead™ Enricher Accessories Kit (4453073
•SOLiD™ EZ Bead™ Enricher Column
•SOLiD™ EZ Bead™ Enricher Input Tube
•SOLiD™ EZ Bead™ Enricher Bead Output Tube
•SOLiD™ EZ Bead™ Enricher QC Output Tube
•SOLiD™ EZ Bead™ Enricher Hyb Tube
•SOLiD™ EZ Bead™ Enricher Capture Oligo Tube
•SOLiD™ EZ Bead™ Enricher Release Tube
•SOLiD™ EZ Bead™ Enricher Capture Bead Tube
•SOLiD™ EZ Bead™ Enricher Unenriched Bead Bottle
•SOLiD™ EZ Bead™ Enricher Filter
•SOLiD™ EZ Bead™ Sipping Assembly
SOLiD™ Pre Deposition Kit (4452805)
•10✕ Terminal Transferase Buffer
•10✕ Cobalt Chloride
•Terminal Transferase
•Bead Linker
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™ Amplifier E20 Accessories Kit
•SOLiD™ EZ Bead™ Enricher E20 Reagent Kit
•SOLiD™ EZ Bead™ Enricher Buffer Kit
•SOLiD™ EZ Bead™ Enricher Accessories Kit

 § Kit contains 2-butanol.
 *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™ Enricher E80 Reagent Kit (4452725)Box 1:
•SOLiD™ EZ Bead™ Enricher E80 – Capture Beads

Box 2:

•SOLiD™ EZ Bead™ Enricher E80 – Capture Oligo
SOLiD™ EZ Bead™ Enricher Buffer Kit (4444140)Box 1:
•SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 2
•SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 3
•SOLiD™ EZ Bead™ Enricher Buffer Kit – Rinse Buffer
•SOLiD™ EZ Bead™ Enricher Buffer Kit – Hyb Buffer

Box 2:

•SOLiD™ EZ Bead™ Enricher Buffer Kit – Capture Buffer
•SOLiD™ EZ Bead™ Enricher Buffer Kit – Release Buffer
•SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1
•SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1

Additive
Box 3:

•SOLiD™ EZ Bead™ Enricher Buffer Kit – Butanol§
SOLiD™ EZ Bead™ Enricher Accessories Kit (4453073
•SOLiD™ EZ Bead™ Enricher Column
•SOLiD™ EZ Bead™ Enricher Input Tube
•SOLiD™ EZ Bead™ Enricher Bead Output Tube
•SOLiD™ EZ Bead™ Enricher QC Output Tube
•SOLiD™ EZ Bead™ Enricher Hyb Tube
•SOLiD™ EZ Bead™ Enricher Capture Oligo Tube
•SOLiD™ EZ Bead™ Enricher Release Tube
•SOLiD™ EZ Bead™ Enricher Capture Bead Tube
•SOLiD™ EZ Bead™ Enricher Unenriched Bead Bottle
•SOLiD™ EZ Bead™ Enricher Filter
•SOLiD™ EZ Bead™ Sipping Assembly
SOLiD™ Pre Deposition Kit (4452805)
•10✕ Terminal Transferase Buffer
•10✕ Cobalt Chloride
•Terminal Transferase
•Bead Linker
SOLiD™ EZ Bead™ E10 System Consumables, 4-pack (4453095)*
•SOLiD™ EZ Bead™ Emulsifier E80 Reagent Kit
•SOLiD™ EZ Bead™ Emulsifier E80 Accessories Kit
•SOLiD™ EZ Bead™ Amplifier E80 Accessories Kit
•SOLiD™ EZ Bead™ Enricher E80 Reagent Kit
•SOLiD™ EZ Bead™ Enricher Buffer Kit
•SOLiD™ EZ Bead™ Enricher Accessories Kit

 § Kit contains 2-butanol.
 *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 Consumables

Item Source
Nuclease-free Water (1 L) Applied Biosystems AM9932
Isopropyl alcohol Sigma-Aldrich I9516

[править] Before you begin

• Prepare an emulsion of the DNA library and P1 Beads with the SOLiD™ EZ Bead™ Emulsifier
• Amplify the library template onto P1 beads using the SOLiD™ EZ Bead™ Amplifier
• Install the Enricher in a different location than the location used to prepare emulsions containing library template and P1 Beads. Emulsification and enrichment in different locations prevents contamination of the sample. Do not install the Enricher and the Emulsifier in the same room.
• Store unlabeled cleaning racks A and B where they can be routinely used with the Enricher. Like the waste container, the cleaning racks are required for normal operation of the Enricher.

[править] Prepare and install the reagents

NB! You can thaw the SOLiD™ EZ Bead™ Capture Oligo on ice as you prepare and install reagents on the instrument

  1. Ensure that the Enricher has been properly cleaned or primed (Clean the Enricher)
    Importent After the Enricher has been run, always clean the instrument before starting another enrichment run.

    File:Solid EZ Bead Enricher Fig1.png|thumb|left|A - reagent Rack A; B - Reagent Rack B]]

  2. Empty the waste bottle:
    a. Remove used reagent racks A and B.
    b. Empty then reinstall the waste bottle.
    c. Screw on the cap with attached tubing to the waste bottle.
  3. Install the Enricher Buffer Kit – Butanol:
    a. Unscrew the cap with attached tubing of the previously installed bottle of butanol.
    b. Unscrew the cap of a new bottle of butanol.
    c. Replace the previously installed bottle of butanol with the new bottle of butanol in the labelled holder of the Enricher (see Figure 1).
    d. Screw the cap with the attached tubing to the new bottle of butanol so that the cap seals the bottle.
  4. Install reagent rack A on the Enricher:
    a. Uncap the bottles on a rack.
    b. Insert the rack on the right top railing (see Figure 1).
    c. Slide the rack until the rack is fully into the instrument and the rack stops.
  5. Remove all used plastic consumables, if necessary.
  6. Prepare the SOLiD™ EZ Bead™ Enricher Capture Oligo:
    a. Thaw the Capture Oligo on ice.
    b. Transfer 1 mL of Capture Buffer (in the SOLiD™ EZ Bead™ Enricher Buffer Kit) from bottle B1 (pink label) to the SOLiD™ EZ Bead™ Enricher Capture Oligo Tube (F lime-green tube).
    c. Add 28 µL of Capture Oligo to the Capture Buffer in the Capture Oligo Tube (F lime-green tube).
    d. Cap the Capture Oligo Tube and gently swirl the tube to mix the solution.
    e. Install the Capture Oligo Tube into the labelled holder on the Enricher (see Figure 1).
    f. Connect the tubing to the Luer-Lok® of the Capture Oligo Tube (F lime-green tube).
  7. Pour the Buffer Kit – CS Solution 1 Additive into bottle B4 (yellow label), containing CS Solution 1, in the SOLiD™ EZ Bead™ Enricher Buffer Kit (see Figure 2 and Table 1).
    File:Example.jpg

     Table 1

    Rack Position Reagent
    A1SOLiD™ EZ Bead™ Enricher Buffer Kit – Rinse Buffer
    A2 SOLiD™ EZ Bead™ Enricher Buffer Kit – Hyb Buffer
    A3 SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 2
    A4 SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 3
    B1 SOLiD™ EZ Bead™ Enricher Buffer Kit – Capture Buffer
    B2 SOLiD™ EZ Bead™ Enricher Buffer Kit – Release Buffer
    B3
    B4 SOLiD™ EZ Bead™ Enricher Buffer Kit – CS Solution 1
    B5
    B6


  8. Ensure that all the bottles in Reagent Rack B are tightly capped, then mix the reagents in the bottles by gently swirling reagent rack B for 10seconds.
  9. Install reagent rack B on the Enricher:
    a. Uncap the bottles on the rack.
    b. Insert the rack on the left top railing (see Figure 1)
    c. Slide the rack until the rack is slid fully into the instrument and the rack stops.
  10. Install the tubes as listed below (see Table 2):
    a. Unscrew the tubing from the cleaning manifold.
    b. Connect the tubing to the designated tubes and bottles (see Figure 1 and Table 2). Match the numbers/colors on the tubing to the numbers/colors on the tubes and bottles.
    c. Secure the connections with a slight, finger-tight, twist. Do not over-tighten the connections.

    Table 2 Ensure that each tube and bottle listed below is firmly seated at the bottom of its assigned location

    Tubes to installMatching numbers and Tubes to install colors on the tubing and tubes
    __ SOLiD™ EZ Bead™ Enricher QC Output Tube5 Yellow
    __ SOLiD™ EZ Bead™ Output Tube 3 Red
    __ SOLiD™ EZ Bead™ Enricher Unenriched Bead Bottle4 Black
    __ SOLiD™ EZ Bead™ Enricher Release Tube 11 Purple
    __ SOLiD™ EZ Bead™ Emulsifier Hyb Tube 10 Tan
    _ Connector on Cleaning Manifold Connect to any other connector on the Cleaning Manifold


  11. Install the bottle of SOLiD™ EZ Bead™ Enricher Capture Beads in the SOLiD™ EZ Bead™ Enricher Capture Bead Tube (9, pink) on the thermomixer:
    a. Unscrew the cap of a new bottle of Capture Beads (stored at 4°C). Ensure that you are using the correct bottle of Capture beads for E10, E20 or E80 scale of enrichment.
    b. Unscrew the cap that has a new SOLiD™ EZ Bead™ Sipping Assembly
    c. Screw the new Sipping Assembly on to the new bottle of Capture Beads. Discard the used bottle that originally contained the new Sipping Assembly.
    d. Place the new bottle of capture beads with the new Sipping Assembly in the labelled location in the Emulsifier (see Figure 1).
    e. Connect the tubing to the Sipping Assembly.
  12. Recover the emulsion:
    a. Tare the SOLiD™ EZ Bead™ Enricher Input Tube with its cap assembly.
    b. Unscrew the cap from the pouch nozzle, and unscrew the cap assembly from the Input Tube.
    c. Pour the entire emulsion from the SOLiD™ EZ Bead™ Amplifier Pouch into the Input Tube.
    d. Screw the cap onto the pouch nozzle.
    e. Lay the pouch flat on a bench top. With a MicroAmp® Adhesive Film Applicator, push the remaining emulsion toward the pouch nozzle.
    f. Uncap the pouch nozzle, then pour the remaining emulsion into the Input Tube.
    g. Screw the cap assembly on the Input Tube.
    h. Screw the cap onto the pouch nozzle, then dispose of the pouch into the appropriate waste container.
  13. Weigh the emulsion in the tared, SOLiD EZ Bead Enricher Input Tube with its cap assembly:
    a. Record the weight of the emulsion for later use: _____________ g.
    b. Follow the table below:

    If the weight of the emulsion is within range...Then...
    E10: 13g to 18g Proceed with step 14
    E20: 22g to 27g
    E80: 100g to 118g
    Outside the ranges aboveDo not proceed with the enrichment run. Contact your Applied Biosystems Field Applications Specialist.


  14. Place the Enricher Input Tube in the labelled location on the Enricher, then attach the tubing to the Input Tube.
  15. Install the Enricher Column (see Figure 1):
    a. Disconnect the cleaning line from the base of the Enricher Column, then connect the cleaning line to any port on the Cleaning Manifold.
    b. Unlock the clamp, if necessary.
    c. Connect the tubing to the top of a new Enricher Column (see Figure ).
    d. Push the male fitting at the bottom of the Enricher column into bottom female connection (Figure ).
    e. Clamp and lock the column.
    File:Example.jpg
  16. Install a Enricher Filter by opening the vibrator stage:
    a. Unscrew the latch, then open the vibrator stage.
    b. Push and twist finger-tight the top connector to the female Luer-Lok™ fitting of the top of the filter.
    c. Push the male fitting on the bottom of the filter into the female Luer-Lok ™ connection on the stage (see Figure ).
    d. Clamp then screw the latch to secure the filter to the vibrator stage (see Figure ).
    File:Example.jpg
  17. Double-check the tubing:
    • Correct connections? Match the color-coded tags on the tubing with the color-coded labels on the tubes and columns.
    • Kinks or tangles? If so, straighten the tubing.
    • Connections tight? Ensure that the top and bottom connections to the Enricher Filter and to the Enricher Column are secure with a slight finger-tight twist. Do not over-tighten the connections.

[править] Run the Enricher

[править] Run the Enricher using the control window

You can run the Enricher overnight or over the weekend. The enriched, unenriched, and QC Output beads can remain at room temperature, but the beads should be stored be at 4°C after the weekend.

NB! Do not open either door of the Enricher during a run. If a door error occurs, the sample cannot be recovered. If the sample is lost because of a door error, follow the procedure Clean the SOLiD™ EZ Bead™ Enricher. Then follow the EZ Bead emulsion process described in the Applied Biosystems SOLiD™ EZ Bead™ Emulsifier Getting Started Guide.

  1. Ensure that the green “POWER” on the front panel is lit (see Figure). If the green “POWER” status is not lit, toggle the power switch to the on position on the back instrument panel, wait until the current run is complete.
  2. Open the Enricher software by double-clicking the icon . The Enricher control window displays (see Figure)
  3. Click the scale-selection button to select the scale in the control window ((E10, E20, E80, or E120; see Figure).
  4. Enter the weight of the emulsion in grams (g).
    IMPORTANT! The emulsion weight must be within the range that is shown in the control window. If the weight of the emulsion is not within the specified range for the scale of run, do not start the enrichment run. Contact your Applied Biosystems Field Applications Specialist.
  5. Click the start-run button . A pre-run checklist displays.
  6. Complete and check off each item in the checklist (see Figure). You cannot proceed with the run until all items are checked off. If necessary, you can exit the checklist by clicking the stop button. After you complete the checklist, ensure that the Enricher doors are closed. Click the check button at the bottom of the checklist to begin the run. After the run starts, the yellow “IN USE” on the front status panel is lit.
    The run time for all scales is about 4hours for the E10, E20, and E80 scales. After the run is complete, the Enricher stops automatically. The green “POWER” status on the front panel is lit.
    IMPORTANT! Do not close the laptop or put the laptop into hibernation mode. Closing the laptop or putting the laptop into hibernation mode can affect communication between the software and the instrument during a run.

    IMPORTANT! Only stop the run if the Enricher malfunctions. If the run is stopped, the sample cannot be recovered. After the instrument stops the run, troubleshoot if necessary, then proceed to Clean the SOLiD™ EZ Bead™ Enricher. After the instrument has been properly cleaned, follow the EZ Bead emulsion process described in the Applied Biosystems SOLiD™ EZ Bead™ Emulsifier Getting Started Guide.

    IMPORTANT! To stop the run, press the stop button. The control window is lit red and displays, “Run aborted”. The red “ERROR” on the control window is lit.


[править] Concentrate and store the samples

IMPORTANT! Do not store the samples in the Enricher Bead Output Tube, the Enricher Unenriched Bead Bottle, or in the SOLiD™ EZ Bead™ QC Output Tube. These tubes are not air-tight, so the samples can leak or evaporate.
  1. Remove the Enricher Bead Output Tube, the SOLiD™ EZ Bead™ QC Output Tube, and the Enricher Unenriched Bead Bottle from the Enricher. The Unenriched Bead Bottle contains a large volume of beads, which is normal.
  2. Immediately concentrate and store the samples.

[править] Concentrate and store the Bead output

  1. Place a 2-mL DNA LoBind Tube into a magnetic rack.
  2. In the Emulsifier Bead Output Tube, pipet the bead solution up and down to resuspend the beads.
  3. Immediately pipet 2 mL of the bead output solution into the 2-mL DNA LoBind Tube.
  4. After the solution clears, remove and discard the supernatant.
  5. Repeat step 3. Do not remove the supernatant.
  6. Vortex and pulse-spin the 2-mL DNA LoBind Tube.
  7. Store the bead output at 4°C until use, or proceed to "Modify the 3' ends of the templated beads".

[править] Concentrate and store the QC output

  1. In the Enricher QC Output Tube, pipet the solution up and down to resuspend the beads.
  2. Pipet all of the bead output solution (1 mL) into a 1.5-mL DNA LoBind Tube.
  3. Store the QC output at 4°C until it is ready to be used.

[править] Concentrate and store the unenriched beads

  1. Swirl the Enricher Unenriched Bead bottle until the beads are resuspended.
  2. Pour the unenriched beads into a 50-mL conical tube.
  3. Centrifuge the 50-mL tube for 5 minutes at 1462× g.
  4. Discard all but 2 mL of the supernatant.
  5. Vortex to resuspend the pellet, then transfer the bead suspension to a 2-mL DNA LoBind Tube.
  6. Store the unenriched beads at 4°C until it is ready to be used.



[править] Modify the 3' ends of the templated beads

NB! Preparation guide for the scale E80

Extend the 3' ends with Terminal Transferase and Bead Linker

  1. If the P2-enriched beads have been stored overnight or longer, sonicate the beads using the Covalent Declump 3 program on the Covaris® S2 System. Pulse-spin the beads.
  2. Prepare 2.4mL of 1x Terminal Transferase Reaction Buffer according to Table:
    Component Volume per reaction (µL)
    10x Terminal Transferase Buffer 264
    10x Cobalt Chloride 264
    Nuclease-free Water 1872
    Total 2400
    Note: The 1x Terminal Transferase Reaction Buffer should be clear. If the solution becomes colored, discard then prepare fresh buffer using a new lot of material.

  3. Add 4 µL of 50 mM Bead Linker to 196µL of 1x Low TE Buffer to prepare a 1mM Bead Linker solution.
  4. Place the tube of P2-enriched beads in a magnetic rack for at least 1minute. After the supernatant clears, remove and discard the supernatant.
  5. Resuspend the beads in 300 µL of 1x Terminal Transferase Reaction Buffer, then transfer the beads to a 2.0-mL DNA LoBind Tube.
  6. Place the tube in a magnetic rack for at least 1minute. After the supernatant clears, remove and discard the supernatant.
  7. Resuspend the beads in 300 µL of 1x Terminal Transferase Reaction Buffer.
  8. Repeat step 6.
  9. Resuspend the beads in 1424µL of 1x Terminal Transferase Reaction Buffer.
  10. Add 160µL of 1mM Bead Linker solution to the tube.
  11. Transfer 792µL of bead solution to a new 2.0-mL DNA LoBind Tube.
  12. Sonicate the beads using the Covalent Declump 3 program on the Covaris® S2 System. Pulse-spin the beads.
  13. Add 8 µL of Terminal Transferase (20 U/µL) to each tube, vortex, then pulse-spin the beads.
  14. Seal the tubes with Parafilm, then place the tubes on a rotator and rotate for 2hours at 37°C.
  15. Pulse-spin the tubes, then pool the beads in one DNA LoBind Tube.
  16. Place the tube in a magnetic rack for at least 1minute. After the supernatant clears, remove and discard the supernatant.
  17. Resuspend the beads in 400 µL of 1x TEX Buffer.
  18. Repeat step 16.
  19. Resuspend the beads in 400 µL of 1x TEX Buffer.

STOPPING POINT Store the templated beads at 4°C in 1x TEX Buffer, or proceed to “Prepare and install slides and reagents” in the 5500 Series SOLiD™ Sequencers Operation Guide.



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