SOLiD_TM System 2.0 UB_miniscale ePCR_4405787A_online_final2_082708

User Bulletin
Applied Biosystems SOLiD™ System 2.0
September 2008 SUBJECT:Important changes to templated bead preparation and a new mini-scale ePCR protocol
In this user
bulletin Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 What is new. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Templated bead preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Definitions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Good practices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX® Tube Drive from IKA®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Mini-scale templated bead preparation: emulsion break and bead wash . . . . . . . . 14 Mini-scale templated bead preparation: emulsion break (standard method) and bead wash. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Mini-scale templated bead preparation: enrichment. . . . . . . . . . . . . . . . . . . . . . . . 18 Mini-scale templated bead preparation: 3′ end modification. . . . . . . . . . . . . . . . . 23 Required Applied Biosystems reagent kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Alternative emulsion break procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Set up the Xstream pipettor for the mini-scale templated bead procedure. . . . . . . 28 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
User Bulletin Applied Biosystems SOLiD™ System 2.0
Overview
This user bulletin describes changes to the templated bead preparation and a new
mini-scale emulsion method for templated bead preparation.
Note:For safety and biohazard guidelines, refer to the Applied Biosystems SOLiD™
System 2.0 User Guide preface and “Safety” appendix. For the MSDS of any
chemical not distributed by Applied Biosystems, contact the chemical manufacturer.
Before handling any chemicals, refer to the MSDS provided by the manufacturer and
observe all relevant precautions.
What is new
Mini-scale emulsion PCR The mini-scale protocol is used to sequence smaller genomes or sequencing samples. The procedure uses half the amount of aqueous phase reagents, SOLiD™ P1 DNA beads and enrichment beads as the standard protocol.
Cut Combitip
Plus tips For both the full- and mini-scale emulsions, the pipette tip that is used to dispense the emulsion in the 96-well PCR plate has been changed. A cut Eppendorf Combitip Plus tip attached to a manual repipettor (Eppendorf Repeater Plus Pipette,
PN022260201) is recommended. The wider bore of this tip reduces the shearing force and helps maintain the emulsion droplet size when dispensing the emulsion into the 96-well plate.
Alternative procedure to
break the
emulsion For both the full- and mini-scale emulsions, the steps in emulsion break have been streamlined by replacing manual pipetting with an adaptor plate. After the PCR reaction is complete, a metal adaptor plate and reservoir are placed over the 96-well plate and centrifuged, forcing the emulsion in each well into a single reservoir.
Single-tube enrichment For both the full- and mini-scale emulsions, the enrichment step has been simplified. Instead of transferring the bead mixture (enrichment and templated beads) into multiple tubes containing 60% glycerol, the bead mixture is transferred and processed in a single tube. This protocol requires a larger DNA LoBind tube (Eppendorf 2.0-mL LoBind Tube, PN022431048).
Templated bead preparation
Templated bead preparation
Two templated-bead preparation procedures are available to SOLiD ™ System 2.0
users: full-scale and mini-scale . The full-scale procedure is used to sequence
complex genomes (refer to the SOLiD ™ System 2.0 User Guide , Chapter 3). The
mini-scale procedure is used to sequence less complex genomes or ligated PCR
products (see “Mini-scale templated bead preparation” on page 7). Each templated-
bead procedure is written for one IKA ®-based ePCR reaction.
The full-scale emulsion is seeded with 1.6billion beads, and the mini-scale emulsion
is seeded with 800 million beads. The table below provides ranges of expected yields
of P2-enriched beads after you prepare either emulsion (refer to the SOLiD ™ System
2.0 User Guide , Chapter 4, “Overview” to determine how many ePCR reactions are
required for the type of run desired):
Y ou perform ePCR when you know the template concentration that gives the best
sequencing results (the “optimal titration point ”). Y ou can find the optimal titration
point by performing library titration analysis (refer to the SOLiD ™ System 2.0 User
Guide , Chapter 4). Y ou can determine the concentration by performing two separate
ePCR reactions at 0.05 pg/µL and 0.1 pg/µL template followed by a titration/QC run.
Both the full-scale and mini-scale procedures are suitable to prepare templated beads
for library titration analysis, but the mini-scale procedure uses less reagents. Emulsion PCR or bead
deposition steps
Full-scale yields Mini-scale yields Isolation of P2-enriched
Beads
150 to 300 million P2-enriched beads 75 to 150 million P2-enriched beads Bead deposition ≥75 to 150 million
P2-enriched beads ≥38 to 75 million P2-enriched beads
User Bulletin Applied Biosystems SOLiD™ System 2.0
Definitions
SOLiD™ P1 DNA
Beads
Bead with P1 adaptor attached.
ePCR Emulsion PCR.
Templated beads SOLiD™ P1 DNA Beads with PCR template attached.
P2-enriched
beads
Enriched templated beads.
Good practices
Resuspend the
beads Y ou can resuspend the beads in one of two ways:
•Gently pipette the solution up and down until the beads are suspended. Y ou can minimize the amount of beads that stick on the inside of the pipette tip by using
a slower speed to aspirate and expel the solution.
•V ortex the solution until all the beads are suspended. Place the beads in a picofuge and pulse-spin for a few seconds to bring down any beads that are
stuck on the walls of the tube. Do not overspin the beads; otherwise, the beads aggregate into a pellet.
Remove the supernatant Use a pipette to carefully remove the supernatant from the tube without disturbing the beads.
Sonicate the
beads Place the tube containing the beads in the tube holder, and place the tube holder in the Covaris™ S2 System. Run the appropriate program.
IMPORTANT!To ensure optimal sonication, use the appropriate adaptor with the Covaris™ S2 System. For sample volumes ≤ 200 µL, use a 0.65-mL tube and adaptor. For sample volumes > 200 µL, use a 1.5-mL tube and adaptor. Place the tube collar at the indicator line of the adaptor. Perform required maintenance as described in the SOLiD™ System 2.0 User Guide, Appendix B.
Transfer the
beads If beads remain in the original tube after transfer, you can use a small additional volume of the appropriate buffer to recover the remaining beads. Do not exceed a total volume of 1.3mL for a 1.5-mL microcentrifuge or LoBind tube.
Store the beads Store the SOLiD™ P1 DNA Beads at 4 °C in 1✕ TEX Buffer.
IMPORTANT!Do not freeze SOLiD™ P1 DNA Beads or templated beads.
Good practices
Adjust the microcentrifuge speed and times Adjust microcentrifuge speeds and times according to the g-forces specified in the protocols. Applied Biosystems recommends the Eppendorf 5417R tabletop microcentrifuge.
Use 0.5-, 1.5- and
2.0-mL LoBind
Tubes
All steps requiring 0.5-, 1.5-, or 2.0-mL tubes must use Eppendorf LoBind tubes.
User Bulletin Applied Biosystems SOLiD™ System 2.0 Workflow
Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX® Tube Drive from IKA®Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX® Tube Drive from IKA®
For the following hazards, see the complete safety alert descriptions in Appendix B,
“Safety” on page29:
CAUTION! CHEMICAL HAZARDS. Emulsion Stabilizer 1, Emulsion
Stabilizer 2, Emulsion Oil, AmpliTaq Gold® DNA Polymerase, UP.
W ARNING! CHEMICAL HAZARDS. 10✕ PCR Buffer, Magnesium
Chloride.
Prepare the oil
phase 1.Follow the steps carefully below to prepare the oil phase in a BD Falcon™
50-mL polypropylene conical tube (PN 352070) using the following
components and volumes:
IMPORTANT!Syringes are required to accurately measure viscous reagents. Be very careful while using a syringe so that you do not cause the reagent tube to overflow! Draw the volume very slowly from the reagent bottle to ensure that no air bubbles are trapped within the syringe. A best practice is to draw some
reagent into the syringe, dispense the entire reagent back to the reagent bottle, then draw the correct volume of reagent.
e a 3-mL syringe to dispense 1.8mL of Emulsion Stabilizer 1 into the
50-mL conical tube.
e a 1-mL syringe to dispense 400µL of Emulsion Stabilizer 2 very
slowly into the 50-mL tube.
c.Pour the Emulsion Oil (approximately 37.8mL) into the tube that has the
Emulsion Stabilizer 1 and Emulsion Stabilizer 2 so that the final volume is
40mL.
2.After the reagents are combined, cap the tube, then vortex until all Emulsion
Stabilizer 1 and Emulsion Stabilizer 2 are incorporated into the Emulsion oil.
3.Allow the mixture to degas for a minimum of 20minutes while you prepare the
aqueous phase (see “Prepare the aqueous phase” on page8). To degas, place the mixture in a conical tube rack and slightly unscrew the conical tube cap.
Component Volume
Emulsion Stabilizer 1 1.8 mL
Emulsion Stabilizer 2400 µL
Emulsion Oil37.8 mL
Total40 mL
User Bulletin Applied Biosystems SOLiD™ System 2.0
e a 10-mL syringe to dispense 9mL of oil phase to a new SOLiD™ ePCR
Tube, then cap the tube.
IMPORTANT!Use of a syringe to dispense the oil phase is critical to proper
emulsion formation.
IMPORTANT!The oil phase must be used within 1week of preparation. Before
using stored oil phase, thoroughly vortex and degas the solution for 20minutes.
Prepare the aqueous phase IMPORTANT!Use pipette tips with filters to minimize contamination when you prepare the aqueous phase.
1.Dilute the ePCR Primer 1 to prepare a 10-µM working stock solution:
a.Add 2µL of ePCR Primer 1 to 18µL of 1✕ Low TE buffer. If you prepare
two library titrations, double the quantities to 4µL of ePCR Primer 1 in
36µL of 1✕ Low TE Buffer.
b.Mix well.
e 1✕ Low TE Buffer and LoBind tubes to prepare a dilution of the library
template to a final concentration of 50pg/µL. Dilute only enough template for the desired number of emulsions. Do not freeze-thaw dilutions of the library more than 3 to 4 times.
IMPORTANT!Stock solutions and dilutions of libraries should be stored at
−20ºC. Libraries should be stored in a freezer at a concentration of 5 ng/µL or greater.
Note:When you prepare dilutions of the library from the stock solution, always dilute with 1✕ Low TE Buffer. When you prepare a dilution, perform a serial dilution of the library to accurately obtain the desired library concentration. For example, perform a 5✕ dilution from 5ng/µL to 1 ng/µL, then perform a 20✕dilution from 1 ng/µL to 50pg/µL.
IMPORTANT!Follow standard molecular biology practices to minimize cross- contamination between library preparations.
3.Select the appropriate titration point, then prepare the aqueous phase by
combining the following reagents in a Nalgene wide-mouth jar according to the table below.
IMPORTANT!New library preparations may require that you determine the best concentration for a sequencing run. Y ou determine the concentration by
performing two separate ePCR reactions at the concentrations of 0.05pg/µL and
0.1pg/µL template followed by a titration/QC run (refer to the Applied
Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX® Tube Drive from IKA®Biosystems SOLiD™ System 2.0 User Guide, Chapter 4). Most libraries that are
run on the ULTRA-TURRAX® Tube Drive from IKA® titrate between titration
points
1 and
2 in the table below.
Note:V olumes below are for a single IKA®-based ePCR reaction to fill a
96-well plate.
4.Keep the aqueous phase on ice until ready to use.
Prepare the SOLiD™ P1 DNA
beads IMPORTANT!The procedures that follow are optimized for the Covaris™ S2 System, which must be specially adapted to prepare beads for the Applied Biosystems SOLiD™ System. Do not use the Covaris™ S1 sonicator or an unadapted Covaris™ S2 System for bead preparation for use with the SOLiD™ System. For more information, contact an Applied Biosystems SOLiD™ System applications specialist.
1.Thoroughly vortex one tube of SOLiD™ P1 DNA Beads. Ensure that any beads
stuck to the cap are washed down by inverting the tube at least once during
vortexing.
2.Place the tube in a picofuge and pulse-spin for a few seconds to bring down any
beads stuck on the walls of the tube.
3.Place the tube of beads in the magnetic rack for 1minute, then remove the
supernatant from the tube.
Component Final
concentration‡
‡The final concentration is based on a total volume of 2800µL, which includes 2720µL of liquid components and 80µL of beads.
Titration 1
(0.05 pg/µL
template)
140pg total
Titration 2
(0.1 pg/µL
template)
280pg total
Volume per reaction (µL)§
§Volumes below are for a single IKA®-based ePCR reaction to fill a
96-well plate.
10✕ PCR Buffer1✕280280
dNTP Mix (100 mM mix
comprised of 25 mM each
dATP, dTTP, dCTP, dGTP)
14 mM (3.5 mM
each
deoxynucleotide)
392392
Magnesium Chloride (1 M)25 mM7070
ePCR Primer 1 (10µM
working stock solution)
40 nM11.211.2
ePCR Primer 2 (500 µM) 3 µM16.816.8
T emplate (50pg/µL)0.05 or 0.1pg/µL 2.8 5.6 Nuclease-free water N/A1647.21644.4 AmpliTaq Gold® DNA
Polymerase, UP (5U/µL)
0.54 U/µL
(1500/titration)
300300
T otal N/A27202720
User Bulletin Applied Biosystems SOLiD™ System 2.0
4.Resuspend the SOLiD™ P1 DNA Beads in 200µL of Bead Block Solution.
V ortex to ensure that all beads are suspended, then place the tube in a picofuge
and pulse-spin for a few seconds to bring down any beads that are stuck on the
walls of the tube.
IMPORTANT!Keep Bead Block Solution at 4 °C until ready for use.
5.Sonicate the beads using the Bead Block Declump program on the Covaris™ S2
System (refer to the SOLiD™ System 2.0 User Guide, Appendix B, for program
conditions).
IMPORTANT!Ensure that the Covaris™ S2 System is degassed, that no bubbles
are present in the system, and that the instrument and tube are properly aligned
for sonication of beads.
6.Place the tube in a picofuge and pulse-spin for a few seconds to bring down any
beads that are stuck on the walls of the tube.
7.Place the tube of beads in the magnetic rack for 1minute, then remove the
supernatant.
8.Resuspend the SOLiD™ P1 DNA Beads in 200µL of 1✕ TEX Buffer. V ortex to
ensure that all beads are suspended, then place the tube in a picofuge and pulse-
spin for a few seconds to bring down any beads that are stuck on the walls of the
tube.
9.Sonicate the beads using the Covalent Declump 1 program on the Covaris™ S2
System (refer to the SOLiD™ System 2.0 User Guide, Appendix B, for program
conditions).
IMPORTANT!To ensure optimal sonication by the Covaris™ S2 System, for
sample volumes ≤ 200µL use a 0.65-mL tube and adaptor. Place the tube collar
at the indicator line of the adaptor.
10.Place the tube in a picofuge and pulse-spin for a few seconds to bring down any
beads that are stuck on the walls of the tube.
11.If you do not use the beads within 10minutes, repeat steps 9 and 10.
IMPORTANT!Do not add beads to the PCR master mix until immediately ready
to use.
Run the ULTRA-TURRAX® Tube Drive from IKA®1.Place the SOLiD™ ePCR Tube containing 9mL of oil phase onto the ULTRA-
TURRAX®device, then twist the tube to lock it into position (see Figure1 on page12).
2.Add 80µL of the SOLiD™ P1 DNA Beads to the aqueous phase that you
prepared in the previous section.
3.Mix beads and aqueous phase by gently swirling the bottle to ensure that the
beads are uniformly dispersed. To minimize foam, do not vortex.
4.Attach a 10-mL Combitip Plus tip onto the Xstream pipettor.
Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX ® Tube Drive from IKA ®
5.Verify that the Xstream pipettor is set up for mini-scale emulsions (2.80mL
solution):
•Dial Setting: Pip
•Speed (aspirate UP): scale 5 (mid-range)
•Speed (dispense DOWN): scale 1 (slowest)
•Total
volume:
2.80 mL
If necessary, reprogram the Xstream pipettor (see “Set up the Xstream pipettor
for the mini-scale templated bead procedure” on page 28).
6.Draw the entire 2.80mL of aqueous phase and bead mixture into the 10-mL
Combitip Plus tip using the Xstream pipettor.
7.Press the Start button on the ULTRA-TURRAX ® Tube Drive from IKA ® (The
time should be set to 5minutes.) Wait for the instrument's fly wheel to engage
and to reach proper speed.
8.Gently place the Combitip Plus tip into the center sample loading hole in the
ULTRA-TURRAX ® cap.
9.Dispense the aqueous phase and bead mixture into the spinning oil phase. When
the entire volume is dispensed, press the center blue button twice on the pipettor
to empty all contents from the Combitip Plus tip.
Note:The ULTRA-TURRAX ® Tube Drive from IKA ® stops after 5minutes.
10.Remove a 5-mL Combitip Plus tip from its packaging, then cut off its end at the
bevel with a razor blade, as shown below:
11.Attach the cut Combitip Plus tip onto an Eppendorf Repeater Plus Pipette.
12.Gently dispense 100µL of emulsion into each well of a 96-well PCR plate and
seal with MicroAmp ™ Clear Adhesive Film or Thermo Scientific Clear Seal
Diamond Heat Sealing Film.
User Bulletin Applied Biosystems SOLiD ™ System 2.0
Figure 1ULTRA-TURRAX ® Tube Drive from IKA ®-based emulsion procedure:
a. Prepare the aqueous phase and bead mix.
b. Set up the ULTRA-TURRAX ®
Tube Drive from IKA ® with the SOLiD ™ ePCR tube and 9 mL oil phase. c. Set the
Xstream pipettor for mini-scale emulsions. d. Aspirate the aqueous phase.
e. Dispense the aqueous phase
Perform the
ePCR reaction
and inspect the
emulsion 1.Set up the following ePCR conditions on the GeneAmp ® PCR System 9700:a.ePCR thermal cycling program:b.Ramp speed: 9600
c.Reaction volume: 50 µL
2.Place the 96-well plate in a GeneAmp ®
PCR System 9700, then start the run.Stage
Temp (°C)Time Hold
95 5 min 40 cycles 93
15 sec 62
30 sec 72
75 sec Hold
727 min Hold 4∞
Mini-scale templated bead preparation: emulsion PCR (ePCR) with the ULTRA-TURRAX® Tube Drive from IKA®
3.After the ePCR Program runs, inspect the bottom of the reaction plate for beads
that fell out of the emulsion. Beads appear as amber-colored specks at the
bottom of a well.
IMPORTANT!A small number of beads may fall out of emulsion and appear as
small brown flecks at the bottom of a well. Applied Biosystems does not
recommend any further processing of emulsions that have more than three wells
of broken emulsion (aqueous phase appears at the bottom of a well).
(Optional) Store
the plate Store the plate at 4 °C, or proceed to “Mini-scale templated bead preparation: emulsion break and bead wash” on page14.
User Bulletin Applied Biosystems SOLiD™ System 2.0
Mini-scale templated bead preparation: emulsion break and bead wash
The emulsion break uses 2-butanol to remove oil from the emulsified templated
beads after amplification. Next, you wash the beads to remove any residual
2-butanol, oil, and aqueous phase containing PCR reagents.
Methods used to
break the
emulsion Standard method
In the standard method, you use a multi-channel pipettor to add and mix 2-butanol into the emulsion in each well of a 96-well plate. Y ou transfer the solution to a 50-mL reservoir then to a 50-mL tube for processing.
Alternative method
In the alternative method, you place a metal adaptor plate and reservoir over a
96-well plate. Y ou centrifuge the assembly to force the emulsion from each well into a single reservoir. Then you add 2-butanol to the reservoir and transfer the solution to a 50-mL tube for processing (see “Alternative emulsion break procedure” on
page27).
IMPORTANT!To use the alternative method, you need to obtain a plate adaptor from an Applied Biosystems SOLiD™ System field applications specialist.
Mini-scale templated bead preparation: emulsion break (standard method) and bead wash Mini-scale templated bead preparation: emulsion break (standard method) and bead wash
For the following hazards, see the complete safety alert descriptions in Appendix B,
“Safety” on page29:
W ARNING! CHEMICAL HAZARD. 2-Butanol, 1✕ Bead Wash Buffer.
Break the emulsion This protocol is for one emulsion. The emulsion is broken using manual pipetting steps. To break the emulsion using a plate adaptor, see “Alternative emulsion break procedure” on page27.
1.In a fume hood, fill a clean, labeled 50-mL reservoir with 2-butanol.
ing a multi-channel pipettor, transfer 100µL of 2-butanol to each well of the
96-well plate containing the emulsion.
3.Insert the pipette tips into the 2-butanol-emulsion mixture and carefully pipette
up and down 4times to mix the 2-butanol into the emulsion.
4.Transfer all rows of the emulsion mix to a clean 50-mL reservoir.
IMPORTANT!To obtain high bead yields, check the plate to ensure that you transfer all beads from the wells to the reservoir. If beads remain in the plate, rinse the wells with an additional 2-butanol to recover residual beads.
5.Transfer all the emulsion and 2-butanol into a 50-mL conical tube.
6.Rinse the reservoir with additional 2-butanol to ensure all residual beads are
recovered. Use this rinse volume to fill the conical tube to 30mL and discard the excess rinse volume.
7.Cap the tube and vortex to mix the solution.
8.Pellet the templated beads by centrifuging at 2000×g for 5minutes.
Note:Consult the manual specific to your centrifuge or rotor to convert
g-forces to rpm.
9.Gently decant the 2-butanol-oil phase into a waste bottle, With the tube inverted,
place it onto paper towels to drain residual 2-butanol-oil.
10.Wait 5minutes to ensure that all the oil is removed.
IMPORTANT!If the pellet begins to slide out, stop decanting, then remove the 2-butanol using a pipette.
User Bulletin Applied Biosystems SOLiD™ System 2.0
Wash the templated beads
1.Place the 50-mL tube upright in a rack, then add 600µL of 1✕ Bead Wash
Buffer to each pellet of templated beads.
2.Let the pellet soak in 1✕ Bead Wash Buffer for 2minutes.
3.Resuspend the pellet by gently pipetting up and down.
4.Transfer the beads from the 50-mL tube to a 1.5-mL tube.
5.Rinse the bottom of the 50-mL tube with an additional 600µL of 1✕ Bead Wash
Buffer, then transfer the wash to the 1.5-mL LoBind tube.
6.V ortex, then centrifuge the tube at 21,000×g (minimum 14,000×g) for
1minute.
7.Remove the top oil phase with a pipette. Remove as much of the oil at the
meniscus as possible.
8.With a new pipette tip, carefully remove the supernatant and discard.
9.Resuspend the pellet by adding 150µL of 1✕ Bead Wash Buffer to the tube,
then vortex to mix the solution. Pulse-spin the tube to bring down any beads stuck on the walls of the tube. Transfer the mixture into a new 1.5-mL tube
Note:Y ou transfer the templated beads to new tubes because oil residue will adhere to the inside of the tube.
10.Rinse the bottom of the original tube with an additional 150µL of 1✕ Bead
Wash Buffer to ensure that all residual beads are transferred to the new 1.5-mL tube.
11.Add an additional 1mL of 1✕ Bead Wash Buffer. V ortex, then centrifuge at
21,000×g (minimum 14,000×g) for 1 minute.
12.Remove all 1✕ Bead Wash Buffer, then resuspend the beads in 200µL of
1✕ TEX Buffer.
13.Place the tube in a magnetic rack for 1minute, then remove the supernatant.
14.Resuspend the beads in 200 µL of 1✕ TEX Buffer.
(Optional) Store the templated
beads Store the templated beads at 4 °C in 1✕ TEX Buffer, or proceed to “Quantitate the beads”.
Quantitate the
beads Determine the quantity of beads recovered using a NanoDrop™ ND-1000 Spectrophotometer or a hemocytometer. Immediately before quantitation of the beads, sonicate the beads using the Covalent Declump 1 program on the Covaris™ S2 System (refer to the SOLiD™ System 2.0 User Guide, Appendix B, for program conditions).
Note:Applied Biosystems recommends quantitating a 1:10 dilution of the beads when you use a NanoDrop™ ND-1000 Spectrophotometer or 1:100 dilution when you use a hemocytometer.
Mini-scale templated bead preparation: emulsion break (standard method) and bead wash
Using the NanoDrop™ ND-1000 Spectrophotometer
Refer to the SOLiD™ System 2.0 User Guide, Appendix C, for procedures on creating a standard curve and quantitating the templated beads.
1.Create a standard curve, if necessary.
e the NanoDrop™ ND-1000 Spectrophotometer to quantitate the beads.
(Optional) Store
the beads Store the beads at 4 °C in 1✕ TEX Buffer, or proceed to “Mini-scale templated bead preparation: enrichment” on page18.
User Bulletin Applied Biosystems SOLiD™ System 2.0
Mini-scale templated bead preparation: enrichment
For the following hazards, see the complete safety alert descriptions in Appendix B,
“Safety” on
page29:
W ARNING! CHEMICAL HAZARDS. Denaturing Buffer, Denaturant.
CAUTION! CHEMICAL HAZARD. Glycerol, 1✕ Low Salt Binding Buffer.
The enrichment protocol isolates the beads that have full-length extension products after ePCR from beads that lack full-length extension products. Beads with full-length extension products are isolated by oligo hybridization using the sequence of the P2 primer. Both monoclonal and polyclonal beads are enriched using this protocol. Enriched templated beads are called P2-enriched beads.
This protocol is designed to enrich the templated beads derived from one ePCR reaction containing 800 million SOLiD™ P1 DNA Beads.
Prepare the Denaturing Buffer
solution The Denaturing Buffer solution that is prepared below is sufficient for only one emulsion.
1.Transfer 1.8mL of Denaturing Buffer into a new tube that can hold 2mL.
2.Add 200µL of Denaturant to the 1.8mL of Denaturing Buffer.
3.Cap the tube, then vortex to mix well.
IMPORTANT!Prepare the prepared Denaturing Buffer solution fresh weekly.
Prepare 60%
glycerol 1.To a 15-mL centrifuge tube, add 4mL of nuclease-free water using a 10-mL
syringe and 6mL of glycerol using a 3-mL syringe (dispensing 3mL of glycerol twice from the syringe).
IMPORTANT!Dispense the glycerol slowly to ensure that the total volume is dispensed.
2.Cap the tube, then vortex to mix well.
IMPORTANT!Prepare the 60% glycerol fresh weekly.
Prepare the enrichment
beads IMPORTANT!The enrichment beads should be used within 1week of preparation. Store prepared enrichment beads in 1✕ Low Salt Binding Buffer at 4 ºC.
1.V ortex the enrichment beads and immediately transfer 300µL of the enrichment
beads to a 1.5-mL LoBind tube.
2.Centrifuge the beads for 5minutes at 21,000×g (minimum 14,000×g), then
remove the supernatant.
3.Resuspend the beads in 900µL of 1✕ Bind and Wash Buffer.。