emPCR Method Manual – Lib-L SV

emPCR Method Manual – Lib-L SVGS FLX Titanium Series

October 2009 (Rev. Jan 2010)

2 / 14 October 2009 (Rev. Jan 2010)

emPCR Method Manual – Lib-L SV

Workfl ow1.

The emulsion-based clonal amplifi cation (emPCR amplifi cation) of a DNA library sample involves 7 major steps, described in Figure 1. Variants of this procedure exist to accommodate different types of DNA libraries and different number of beads requirements for the experiment. The method described in this manual is for Rapid, Paired End, and cDNA Rapid libraries (Lib-L), and for Small Volume emulsions (SV). This protocol can also be used for processing General libraries (see the GS FLX Titanium General Library Preparation Method Manual, April 2009).

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For life science research only. Not for diagnostic procedures.

3 / 14 October 2009 (Rev. Jan 2010)


Before You Begin2.

If processing sequence capture arrays, refer to the NimbleGen Arrays User’s Guide for amounts of library to use in the emulsion titration.

Sample Handling Strategy for Small Volume Emulsions2.1

Small Volume emulsion samples can be processed either as 16 unique samples, referred to as 1×SVE in this manual, and destined to be loaded in Small regions of a PicoTiterPlate device; or as 8 samples each made of 2 pooled emulsions, referred to as 2×SVE and destined to be loaded in M/S regions; or as 4 samples each made of 4 pooled emulsions, referred to as 4×SVE and destined to be loaded in Medium regions, or as 2 samples each made of 8 pooled emulsions, referred to as 8×SVE and destined to be loaded in Large regions. Figure 2 shows the different sample processing strategies for Small Volume emulsions.

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Before You Begin

4 / 14 October 2009 (Rev. Jan 2010)


Determining the Amount of Library to Use in emPCR Amplifi cation2.2

To clonally amplify the library, the optimal amount of DNA to add to the Capture Beads must be determined.

If the library yield can be predicted from past experience, one can use a pre-determined number of DNA molecules per bead. However, if the DNA library yield is unknown, one can perform a titration. Two titration options are available. The simplest and less time consuming option is the emulsion titration, which consists of using the % of bead enrichment as an indicator of the sequencing quality. This is achieved by performing emPCR amplifi cations on various amounts of library DNA and determining the % of enriched beads. The more involved option is the Sequencing titration, which consists of sequencing the test samples obtained after an emulsion titration.

� For many common purposes, an input of 4 to 6 molecules of library DNA per emPCR bead will generate satisfactory sequencing results.

� Using a pre-determined, fi xed amount of DNA input offers the experienced user the obvious advantage of a faster process. Even if the resulting sequencing Run is not optimal, a signifi cant amount of data will be obtained, without the expense of any optimization.

Emulsion Titration2.2.1

During emulsion titration, various amounts of library are added to a fi xed number of DNA Capture Beads, and carried through the emPCR amplifi cation procedure, including enrichment of the DNA-carrying beads and counting of those enriched beads. The % of enriched beads can be used as an indicator for the quality of the amplifi ed library: the optimal amount of DNA to use in emPCR amplifi cation. The best sequencing results are obtained with approximately 8% Enriched Beads (a range of 5 – 20% Enriched Beads is usually acceptable).

The emulsion titration assay described below requires a GS FLX Titanium SV emPCR Kit (Lib-L) and a GS FLX Titanium emPCR Filters SV 64pcs Kit.

1 Get the 1 × 107 molecules/µl stock of the quantitated DNA library to be tested. Dilute it as follows, depending on the type of library:

a. For General, Rapid or Paired End libraries, dilute 20 µl of library into 30 µl of TE Buffer to make a 4 × 106 molecules/µl solution.

b. For cDNA Rapid libraries, whose yield is typically lower, dilute 20 µl of library into 5 µl of TE Buffer to make a 8 × 106 molecules/µl solution.

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2 Set up four single-tube SV emPCR amplifi cation reactions (2.4 × 106 Capture Beads per emulsion tube), and prepare samples containing the four different amounts of DNA library, as follows:

TubeTarget Molecules per Beads

Volume of diluted libraryGeneral, Rapid or PE cDNA Rapid

1 2 4 1.2 µl

2 4 8 2.4 µl

3 8 16 4.8 µl

4 16 32 9.6 µl

3 Vortex the four tubes for 5 seconds to mix their contents.

4 Perform the rest of the emPCR amplifi cation procedure, using the “Small Volume Emulsion” procedure described in the GS FLX Titanium emPCR Method Manual Lib-L SV, (the present manual).

� The enriched beads are counted at the end of the emPCR amplifi cation procedure.

5 For each amount of DNA input, calculate the Enriched Beads as a percentage of the total number of beads used:

% Enriched Beads =Number of fi nal enriched beads

× 1002.4 × 106 Total input beads

6 For large scale emPCR and sequencing on this library, use the amount of input DNA that gives an enriched bead yield of approximately 8%.

� You may interpolate between the 4 data points. For example, if the results of the emulsion titration are: 2 molecule/bead = 1% Enriched Beads 4 molecules/bead = 3% Enriched Beads 8 molecules/bead = 6% Enriched Beads 16 molecules/bead = 12% Enriched Beads

… then you could interpolate that 10 molecules/bead would yield approximately 8% Enriched Beads.

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� If all four amounts of DNA tested in the emulsion titration assay yield a % Enrichment Beads below (or, more rarely, above) 8%, you may have incorrectly assessed the concentration of the library. If this happens, either repeat the quantitation fl uorometric assay and perform the titration with the corrected results, or simply repeat the emulsion titration with more or less input DNA (e.g. 8, 16, 32 and 64 molecules/bead for a General, Rapid or a Paired End library; this may require a new dilution of the stock library, e.g. to 1.6 × 107 molecules/µl).

� If a poor correlation is observed between the amount of input DNA and the % Enriched beads, the emulsion titration can be repeated with duplicate tubes rather than single tubes, at each amount of DNA.

Before You Begin

5 / 14 October 2009 (Rev. Jan 2010)


Sequencing Titration2.2.2

The sequencing titration described below comprises both emPCR amplifi cation and sequencing reactions. It requires a GS FLX Titanium SV emPCR Kit (Lib-L), a GS FLX Titanium emPCR Filters SV 64pcs Kit, a GS FLX Titanium Sequencing Kit XLR70, and a GS FLX Titanium PicoTiterPlate Kit 70 × 75 (using the small regions “multi-lane” gasket). Also refer to the GS FLX Titanium Sequencing Method Manual.

1 Perform the emulsion titration.

2 Load up to 125,000 beads from each of the 4 amplifi ed library aliquots on a PicoTiterPlate device, using a small regions “multi-lane” gasket.

The lower DNA input reactions (e.g. 2 molecule/bead) may not yield that number of enriched beads. In such a case, load all the beads produced by this reaction. The assay will show a correspondingly low number of reads, which is the correct assessment for a small DNA input.

3 Perform the sequencing procedure. Find the number of totalPassedFiltering reads (for the Library key), using the GS Run Browser or in the 454QualityFilterMetrics.csv (or .txt) fi le generated by the GS Run Processor application (see the Genome Sequencer System Software Manual for details on the GS Run Browser and GS Run Processor software).

4 For large scale emPCR and sequencing on this library, use the amount of input DNA that gives the largest number of totalPassedFiltering reads. A good library should give at least 25,000 totalPassedFiltering reads in the best region., and a > 5% enrichment bead.

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Procedure3.

Preparation of the Reagents and of the Emulsion Oil3.1

Preparation of the Reagents3.1.1

1 Open the emPCR Reagents box and thaw the kit components at room temperature, except the enzymes which should be kept at -15°C to -25°C. Once thawed, vortex for 5 seconds.

2 Vortex and heat the tube of emPCR Additive at 55ºC for 5 minutes to aid dissolving. If a precipitate persists, centrifuge the tube and use the supernatant.

3 Spin all the kit components (including enzymes) in a bench top mini centrifuge for 10 seconds.

4 Return the enzymes to -15°C to -25°C. The other reagents can be left at room temperature.

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Preparation of the Emulsion Oil3.1.2

Rattling sound: If the TissueLyser makes a loud rattling sound, turn it off immediately and make sure that all tubes and adapters are screwed tightly in place.

1 Place up to 8 tubes in the outer row only, of each of the two TissueLyser tube racks.

2 Shake the Emulsion Oil at 25 Hz for 2 minutes.

3 Remove the shaken Emulsion Oil from the TissueLyser.

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Preparation of the Mock Amplifi cation Mix and Pre-Emulsions3.1.3

If not processing a full kit, adjust the volumes accordingly.

1 Dilute 1 ml of 5× Mock Amplifi cation Mix with 4 ml of Molecular Biology Grade Water to make a working solution. Vortex.

2 Add 290 µl of the Mock Amplifi cation Mix working solution to each tube of Emulsion Oil.

3 Invert the tube racks 2 – 3 times to mix, place on the TissueLyser in outer row only, and secure fi rmly.

4 Shake in the TissueLyser at 25 Hz for 5 minutes.

5 Remove the emulsions from the TissueLyser.

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Procedure

6 / 14 October 2009 (Rev. Jan 2010)


Preparation of the Live Amplifi cation Mix3.1.4

1 Prepare the Live Amplifi cation Mix for the number of emulsion reactions being made, according to Table 1 A (for General, Rapid and cDNA Rapid libraries) and B (for Paired End libraries). Add the reagents in the order they are listed in the tables.

2 Vortex the Live Amplifi cation Mix for 5 seconds, and store on ice.

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A B

ReagentVolume (µl)

ReagentVolume (µl)

4 Tubes 8 Tubes 16 Tubes 4 Tubes 8 Tubes 16 Tubes

Mol. Bio. Grade Water 280 560 1120 Mol. Bio. Grade Water 320 640 1285

emPCR Additive 360 720 1440 emPCR Additive 360 720 1440

5× Amplifi cation Mix 190 380 760 5× Amplifi cation Mix 190 380 760

Amplifi cation Primer 55 110 220 Amplifi cation Primer 15 30 55

emPCR Enzyme Mix 50 100 200 emPCR Enzyme Mix 50 100 200

PPiase 2 4 8 PPiase 2 4 8

Total: 937 1874 3748 Total: 937 1874 3748

Table 1: Preparation of the Live Amplifi cation Mix. A: for General, Rapid and cDNA Rapid libraries; B: for Paired End libraries.

DNA Library Capture3.2

1 Prepare 1× Capture Bead Wash Buffer TW by mixing 1 ml of 10× Capture Bead Wash Buffer TW with 9 ml of Molecular Biology Grade Water.

2 Vortex the tubes of DNA Capture Beads.

3 Transfer 80 µl of DNA Capture Beads (per reaction) to a clean 1.7 ml tube (up to 1280 µl, or 16 emulsion reactions, per tube; a full SV kit fi ts in a single tube).

4 Pellet the beads in a bench top minifuge, by spinning for 10 seconds, rotating the tube 180°, and spinning again for 10 seconds.

5 Carefully remove and discard the supernatants without disturbing the bead pellets.

6 Wash each tube of beads twice with 1 ml of 1× Capture Bead Wash Buffer TW. Vortex to resuspend the beads, spin, and discard the supernatant after each wash.

7 Distribute the washed DNA Capture Beads into single emulsion aliquot size.

a. Resuspend each pellet of DNA Capture Beads in 640 µl of 1× Capture Bead Wash Buffer TW (or 40 µl times the number of emulsion tubes prepared).

b. Measure the total volume of DNA Capture Beads suspension and distribute them evenly between the appropriate number of 1.7 ml tubes.

c. Pellet the beads as before, remove and discard the supernatant.

8 Thaw an aliquot of the DNA library to be amplifi ed.

� Heat denature DNA prepared with the Rapid and the cDNA Rapid library methods, by running the following program on a thermocycler, with the heated lid on:

95°C for 2 minutes

4°C on hold

9 To each tube of washed DNA Capture Beads, add the correct volume of the DNA library that will provide optimal amplifi cation (e.g. per titration) to the bead pellet.

a. Use the following equation:

µl of library per tube =desired molecules per bead × 2.4 × 106 beads per tube

library concentration (in molecules/µl)

b. If necessary, prepare a dilution of the library such that the volume to be added will be between 1 µl and 10 µl. Repeat heat denaturation when processing DNA processed with the Rapid and the cDNA Rapid library methods.

10 Vortex the tubes for 5 seconds to mix their contents.

11 Add 215 µl of Live Amplifi cation Mix to each tube of captured library mix. Vortex.

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Procedure

7 / 14 October 2009 (Rev. Jan 2010)


Emulsifi cation3.3

1 Pipet the content from each tube of captured library from Section 3.2 into a prepared emulsion tube (from Section 3.1.2).

2 Invert the tube racks 3 times to mix. Place the tubes in the TissueLyser, only in the outer row.

3 Shake in the TissueLyser at 15 Hz for 5 minutes.

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Amplifi cation3.4

Dispensing the Emulsions3.4.1

1 After emulsifi cation, dispense the emPCR amplifi cation mixes into 96-well thermocycler plates, at 100 µl per well; each tube of emulsion will fi ll approximately 10 wells.

� Emulsion in tube lids: Some emulsion may have lodged in the tube lids. You can pipet it up from the lids but DO NOT spin the emulsion tubes in an attempt to reclaim this material as this would risk breaking the emulsions.

� Air bubbles: dislodge any air bubbles that may be present at the bottom of the wells by gently tapping the plate.

2 Cap the wells and make sure that all the wells are properly sealed.

3 Clean up the area of any spilled reaction mix.

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Amplifi cation Reaction3.4.2

1 Bring the plates containing the emulsifi ed amplifi cation reactions to the post-amplifi cation area, place them in a thermocycler, and start the amplifi cation program with the lid set to track within 5°C of the block temperature. The program takes ~6 h to complete.

1× 4 minutes at 94°C

50× 30 seconds at 94°C, 4.5 minutes at 58°C, 30 seconds at 68°C10°C on hold

� Do not freeze the DNA beads: You can leave the amplifi cation reactions at 10°C for up to 16 hours before further processing the samples.

� Emulsion breakage: Check all wells for emulsion breakage (i.e. faint yet distinct layers with clearer middle layer). If the emulsion in any well appears broken, discard the entire well and do not recover the beads from it. See Section 4.2 for pictures of intact and broken emulsions.

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Bead Recovery3.5

Emulsion Collection and Initial Washes3.5.1

1 Retrieve the Enhancing Fluid XT and the Annealing Buffer XT, and keep at room temperature.

2 Screw a 16 gauge blunt, fl at tip needle directly onto the end of a 10 ml syringe. You will need one syringe and blunt needle for each sample you are processing, i.e. up to sixteen syringes for 1×SVE; eight for 2×SVE; four for 4×SVE, and 8×SVE. Up to 4 emulsions can be pooled into a single syringe.

3 Draw the emulsion from each well of a sample into the appropriate number of syringes.� For 1×SVE, expect about 10 wells, or 1 ml total� For 2×SVE, expect about 20 wells, or 2 ml total� For 4×SVE, expect about 40 wells, or 4 ml total� For 8×SVE, expect about 80 wells (40 wells × 2), or 8 ml (4 ml × 2) total

4 Add 100 µl of isopropanol to each well. Mix by pipetting up and down.

5 Draw the isopropanol rinse from the wells of each sample into the same syringe(s).

6 Draw again to collect the material that drained from the walls of the wells.

7 Invert the syringe and draw in 3 ml of air.

8 Remove and discard the blunt needle.

9 Attach to the syringe, without over tightening, a blue fi lter from a GS FLX Titanium emPCR Filters, SV, 64 pcs kit.

10 Place a Kimwipe on top of the vortex platform and vortex the syringe for 5 seconds at maximum speed, with the fi lter pointing down toward the Kimwipe.

11 Gently expel the contents of the syringe through the fi lter, into a waste jar containing bleach.

Pressure buildup: If there is a buildup of pressure in the syringe during the wash, do NOT continue to expel the liquid, but draw air and slowly expel the remaining liquid.

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Procedure

8 / 14 October 2009 (Rev. Jan 2010)


Bead Washes and Recovery3.5.2

Tube requirement: All 1.7 ml tubes must be siliconized.

1 Invert the syringe and draw in 3 ml of air. When processing 4xSVE or 8xSVE, the syringe is nearly full, therefore do not draw any air.

2 Draw 3 – 5 ml of isopropanol into the syringe, without drawing air.

3 Vortex the syringe for 5 seconds at maximum speed, with the fi lter pointing down, onto the Kimwipe.

4 Expel the contents of the syringe through the fi lter, into a waste jar containing bleach.

5 Repeat steps 1 to 4 two more times.


7 Draw 3 ml of Enhancing Fluid XT into the syringe, without drawing air.


9 Expel the contents of the syringe through the fi lter, into a waste jar containing bleach.

10 Repeat steps 5 to 8 two more times.


12 Draw Enhancing Fluid XT into the syringe to the 0.5 ml mark, without drawing additional air. The total volume should be ~800 µl.


14 Promptly invert the syringe and draw in another 3 ml of air.

15 Remove (but keep) the fi lter from the syringe and dispense the bead suspension in a new 1.7 ml collection tube.

16 Spin the 1.7 ml collection tube for 10 seconds, rotate the tube 180°, and spin again for 5 seconds. Remove and discard the supernatant.


18 Place the fi lter back on the syringe, and draw Enhancing Fluid XT into the syringe to the 0.5 ml mark, without drawing additional air.

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20 Promptly invert the syringe and draw in another 3 ml of air.

21 Remove the fi lter from the syringe and dispense the bead suspension in the same 1.7 ml collection tube.

22 When processing 4×SVE or 8×SVE, repeat steps 15 to 21 for a third fi lter wash.

23 Discard the syringe and the fi lter.

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DNA Library Bead Enrichment3.6

� At this point, you will have one 1.7 ml collection tube for each syringe processed (for a full kit, 16 tubes for 1×SVE, 8 tubes for 2×SVE or 4 tubes for 4×SVE and 8×SVE).

� The reagent volumes listed in this section apply to each tube of beads.

Preparation for Indirect Enrichment3.6.1

Hazardous Chemical – Sodium Hydroxide Solution: Sodium hydroxide (present in the Melt Solution) is a highly corrosive chemical that may cause burns if it contacts eyes or skin. Read the Material Safety Data Sheet for handling precautions.

1 Turn on the heating dry-block and set it to 65°C.

2 Prepare the Melt Solution by mixing 125 µl of NaOH (10 N) in 9.875 ml of Molecular Biology Grade Water.

3 Spin-rotate-spin the tubes of beads and discard the supernatant.

4 Add 1 ml of Melt Solution per tube of beads and vortex. Incubate for 2 minutes at room temperature. Spin-rotate-spin and discard the supernatant.

5 Repeat step 4 once.

6 Add 1 ml of Annealing Buffer XT per tube of beads and vortex. Spin-rotate-spin and discard the supernatant.

7 Repeat step 6 once.

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Procedure

9 / 14 October 2009 (Rev. Jan 2010)


8 Optional: To determine the % breaking effi ciency, do the following:

a. Add 200 µl of Annealing Buffer XT and vortex.

b. Count a 3 µl aliquot of the bead suspension. A recovery of over 65% would be typical at this point of the procedure. Calculate the % bead recovery using following equation:

% Bead Recovery =Number of beads recovered

× 100Total input beads*

* 2.4 × 106 for 1×SVE, 4.8 × 106 for 2×SVE, 9.6 × 106 for 4×SVE, or 19.2 × 106 (9.6 × 106 per tube) for 8×SVE

c. Spin-rotate-spin and discard the supernatant.

9 Add 30 µl of Annealing Buffer XT per tube, and the proper amount of Enrichment Primer for the sample type as below, and vortex.

a. 6 µl for 1×SVE

b. 12 µl for 2×SVE

c. 24 µl for 4×SVE and 8×SVE

10 Place the tubes in a heat block at 65°C for 5 minutes, and then promptly cool on ice for 2 minutes.

11 Add 500 µl of Enhancing Fluid XT per tube and vortex. Spin-rotate-spin and discard the supernatant.

12 Repeat step 11.

13 Add 800 µl of Enhancing Fluid XT per tube and vortex.

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Preparation of the Enrichment Beads3.6.2

1 Vortex the tube of brown Enrichment Beads for 1 minute to resuspend its contents completely.

� You will need 20 µl of Enrichment Beads for each 1×SVE sample, 40 µl for 2×SVE, or 80 µl for 4×SVE and 8×SVE.

� If you are not processing a full emPCR Kit (containing 16 emulsion oil tubes), aliquot 20 µl of Enrichment Beads for each emulsion oil tube processed, and store the rest at +2ºC to +8ºC.

2 Pellet the Enrichment Beads using a Magnetic Particle Concentrator (MPC).

3 Discard the supernatant, taking care not to draw off any Enrichment Beads.

4 Add 500 µl of Enhancing Fluid XT and vortex.

5 Pellet the Enrichment Beads using an MPC.

6 Discard the supernatant, taking care not to draw off any Enrichment Beads.

7 Repeat steps 4 to 6.

8 After discarding the supernatant, remove the tube from the MPC.

9 Add 20 µl of Enhancing Fluid XT per emulsion oil tube processed (320 µl if processing a full kit worth), and vortex.

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Procedure

10 / 14 October 2009 (Rev. Jan 2010)


Enrichment of the DNA-Carrying Beads3.6.3

1 Add 20 µl per 1×SVE, 40 µl per 2-SVE, or 80 µl per 4×SVE and 8×SVE of washed Enrichment Beads to each tube of amplifi ed DNA beads (from Section 3.6.1) and vortex.

2 Rotate the tubes on the Labquake, at room temperature (+15 to +25ºC) for 5 minutes.

3 Place the tubes in the MPC, and wait 3 – 5 minutes to pellet the Enrichment Beads.

4 Invert the magnet several times.

5 Carefully discard the supernatant from each tube using a 1000 µl pipette, taking care not to draw off any brown Enrichment Beads.

� Optionally, collect the supernatants containing “anti-enriched” beads for troubleshooting.

6 Wash the beads with Enhancing Fluid XT per tube until there are no visible beads remaining in the supernatants, as follows:

a. Add 500 µl of Enhancing Fluid XT per tube.

b. Remove the tubes from the MPC and vortex them.

c. Place the tubes back into the MPC to pellet the beads on the wall of the tube with the magnet. Invert the MPC.

d. Carefully discard the supernatant from each tube with a 100 µl pipette, taking care not to draw out Enrichment Beads.

e. Repeat 3 to 6 times until white DNA beads are no longer being aspirated.

� Optionally, collect the supernatants of each wash, and spin to monitor when washes are complete.

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Collection of the Enriched DNA Beads3.6.4

1 Remove the tubes of enriched beads (enrichment tubes) from the MPC and resuspend each bead pellet in 700 µl of Melt Solution.

2 Vortex for 5 seconds, and place the enrichment tubes in the MPC until the Enrichment Beads have pelleted.

3 Transfer the supernatants containing enriched DNA beads:

� into a 1.7 ml collection tube, for 1×SVE, 2×SVE, or 4×SVE.

� into a single 1.7 ml collection tube, pooling both supernatants from the same sample, for 8×SVE.

4 Spin-rotate-spin the 1.7 ml collection tube, and discard the supernatant.

5 Add once again 700 µl of Melt Solution to the enrichment tubes.

6 Vortex for 5 seconds, and place the enrichment tubes in the MPC until the Enrichment Beads have pelleted.

7 Transfer the supernatants containing enriched DNA beads into the same 1.7 ml collection tube.

8 Discard the enrichment tubes.

9 Spin-rotate-spin the collection tubes and discard the supernatant.

10 Add 500 µl of Annealing Buffer XT per collection tube and vortex for 5 seconds.

11 Spin-rotate-spin and discard the supernatant.

12 Repeat steps 10 and 11 two times.

13 Resuspend each bead pellet in the proper amount of Annealing Buffer XT for the sample type as below, and vortex.

a. 30 µl for 1×SVE


c. 120 µl for 4×SVE

d. 200 µl for 8×SVE

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Procedure

11 / 14 October 2009 (Rev. Jan 2010)


Sequencing Primer Annealing3.7

1 Add the proper amount of Sequencing Primer for the sample type as below, and vortex.

a. 6 µl for 1×SVE


c. 24 µl for 4×SVE

d. 48 µl for 8×SVE

2 Place the collection tubes in a heat block at 65°C for 5 min, and then promptly on ice for 2 min.

3 Add 500 µl of Annealing Buffer XT per collection tube and vortex for 5 seconds. Spin-rotate-spin, and discard the supernatant.

4 Repeat step 3 two times with 500 µl of Annealing Buffer XT.

5 Add 100 µl of Annealing Buffer XT to each bead pellet and vortex.

6 To determine the % bead enrichment, do the following:

a. Count a 3 µl aliquot of the beads with a particle counter.

b. Calculate the % bead enrichment using the following equation:

% Bead Enrichment =Number of enriched beads

× 100Total input beads*

* 2.4 × 106 for 1×SVE, 4.8 × 106 for 2×SVE, 9.6 × 106 for 4×SVE, or 19.2 × 106 (9.6 × 106 per tube) for 8×SVE

c. Percent enrichment values between 5 and 20% are indicative of libraries that usually yield good sequencing results.

7 Store the beads at +2°C to +8°C and sequence them within two weeks.

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Procedure

12 / 14 October 2009 (Rev. Jan 2010)


Appendix4.

Bead Counter Settings4.1

454 Life Sciences Corporation, a Roche company, recommends the use of the Innovatis CASY TTC, TT, and DT models, as well as the Coulter Counter Multisizer 3, Z2, and Z1 models from Beckman Coulter for the quantitation of amplifi ed DNA library beads obtained from emPCR amplifi cation reactions. Table 2 shows the settings of the CASY models for bead counting. Figure 3 shows the settings of the Coulter Counter Multisizer 3 model and Table 3, the settings of the Coulter Counter Multisizer Z2 or Z1 models.

Parameter

Settings

Dual Thresholds

Upper Size (Tu) 25 µm

Lower Size (Tl) 15 µm

Count Mode Between

Aperture 150 µm

Metered Volume 1.2 ml (400 µl × 3)

Result Type Concentration

Dilution Factor* 3333 (10 ml diluent)

Table 2: Innovatis CASY TTC, TT, and DT models settings for bead counting. * for 3 µl of diluent. Figure 3: Coulter Counter Multisizer 3 settings for bead counting.

Parameter

Settings

Dual Thresholds Single Threshold

Upper Size (Tu) 25 µm –

Lower Size (Tl) 15 µm 15 µm

Count Mode Between Above Tl

Aperture 100 µm 100 µm

Metered Volume 1.0 ml 1.0 ml

Result Type Concentration Concentration

Dilution Factor* 3333 (10 ml diluent) 3333

Table 3: Coulter Counter Z2 or Z1 models settings for bead counting. Dual thresholds are preferable, if available. * for 3 µl of diluent.

Appendix

13 / 14 October 2009 (Rev. Jan 2010)


Pictures of Emulsions4.2

A B

Figure 4: Intact (A) and broken (B) emulsions. Intact emulsions appear as homogeneous suspensions, whereas broken emulsions show distinct layers, and sometimes an air bubble at the bottom of the well.

Appendix

Published by

Roche Diagnostics GmbHRoche Applied Science68298 MannheimGermany

© 2010 Roche Diagnostics All rights reserved.

�0110


For Life Science Research Only. Not for Use in Diagnostic Procedures

454, 454 LIFE SCIENCES, 454 SEQUENCING, GS FLX, GS FLX TITANIUM,

emPCR, PICOTITERPLATE, and PTP are trademarks of Roche.

Other brands or product names are trademarks of their respective holders.

Table of Contents

1. Workfl ow ...........................................................................................................................2

2. Before You Begin ..........................................................................................................3

2.1 Sample Handling Strategy for Small Volume Emulsions ...................................................3

2.2 Determining the Amount of Library to Use in emPCR Amplifi cation...........................4

2.2.1 Emulsion Titration ............................................................................................................................42.2.2 Sequencing Titration .......................................................................................................................5

3. Procedure ..........................................................................................................................5

3.1 Preparation of the Reagents and of the Emulsion Oil ........................................................5

3.1.1 Preparation of the Reagents ........................................................................................................53.1.2 Preparation of the Emulsion Oil ..................................................................................................53.1.3 Preparation of the Mock Amplifi cation Mix and Pre-Emulsions ....................................53.1.4 Preparation of the Live Amplifi cation Mix ..............................................................................6

3.2 DNA Library Capture ......................................................................................................................6

3.3 Emulsifi cation ....................................................................................................................................7

3.4 Amplifi cation ......................................................................................................................................7

3.4.1 Dispensing the Emulsions ............................................................................................................73.4.2 Amplifi cation Reaction ...................................................................................................................7

3.5 Bead Recovery ..................................................................................................................................7

3.5.1 Emulsion Collection and Initial Washes...................................................................................73.5.2 Bead Washes and Recovery ........................................................................................................8

3.6 DNA Library Bead Enrichment ...................................................................................................8

3.6.1 Preparation for Indirect Enrichment..........................................................................................83.6.2 Preparation of the Enrichment Beads ......................................................................................93.6.3 Enrichment of the DNA-Carrying Beads ..............................................................................103.6.4 Collection of the Enriched DNA Beads .................................................................................10

3.7 Sequencing Primer Annealing ..................................................................................................11

4. Appendix ........................................................................................................................ 12

4.1 Bead Counter Settings .................................................................................................................12

4.2 Pictures of Emulsions ...................................................................................................................13

Documents

emPCR Method Manual – Lib-L SV