Thermo Fisher Scientific PrepFiler User Manual

PrepFiler™ Automated Forensic DNA Extraction Kit

USER BULLETIN

Automated DNA Puriﬁcation on the HID EVOlution™ Systems

for use with: HID EVOlution™—Extraction System HID EVOlution™—Combination System

Publication Number MAN0019298

Revision A.0

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019298

Revision Date Description

A.0

22 March 2021 New document.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Trademarks: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. Freedom EVO, EVOware, Te-Shake, and HID EVOlution are trademarks of Tecan Group AG. Microsoft and Windows are trademarks of Microsoft Corporation.

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CHAPTER 1 Product information .................................................. 6

Product description ............................................................. 6

About the HID EVOlution™ systems ................................................ 6

Puriﬁcation run times ........................................................ 7

System components ........................................................ 8

Plate/tube conﬁgurations .................................................... 8

Contents and storage ............................................................ 8

Required materials not supplied ................................................... 9

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CHAPTER 2 Prepare for the automated puriﬁcation run ...................... 11

Perform lysis .................................................................. 11

Before ﬁrst use: Prepare the wash buers ........................................ 11

Before each use: Prepare the magnetic particles .................................. 11

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CHAPTER 3 Set up the robotic workstation .................................... 12

For more information ........................................................... 12

Workﬂow ..................................................................... 13

Perform routine maintenance .................................................... 14

Prepare the system liquid carboy ............................................ 14

Empty the waste carboy .................................................... 14

Tighten the DiTi adapter gold cones .......................................... 14

Run maintenance scripts ................................................... 15

(HID EVOlution™—Combination System only) Set up the carriers and labware .......... 16

Set up the disposable pipette tips ................................................ 19

Terms for pipette tips used on the robotic workstation ......................... 19

Fill DiTi carriers and racks ................................................... 20

Set up reagents on the workstation ............................................... 20

Procedural guidelines ...................................................... 20

Set up the reagents ........................................................ 21

Set up lysate, processing, and elution plates and/or tubes .......................... 23

Select a plate/tube conﬁguration ............................................ 23

Set up the PrepFiler™ Processing Plate ....................................... 24

(If needed) Place barcodes .................................................. 25

PrepFiler Bulletin

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Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

Contents

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Set up lysate and/or eluate plates ............................................ 25

Set up lysate and/or eluate tubes ............................................ 27

Workstation layouts ............................................................ 30

CHAPTER 4 Perform the automated DNA puriﬁcation run .................... 34

For more information ........................................................... 34

Workﬂow ..................................................................... 35

Before you begin ............................................................... 36

Set up and run a script ......................................................... 36

About script ﬁles .......................................................... 36

Select a script ............................................................. 36

Set up sample and reagent information ....................................... 38

Conﬁrm workstation setup and start the run .................................. 44

(If needed) Re-cap the magnetic particles tubes ............................... 46

Record ﬁle information and exit the script ..................................... 47

Complete the run .............................................................. 48

View the qPCR/STR Sample Input and Report ﬁles ................................ 49

About the ﬁles ............................................................ 49

View the ﬁles .............................................................. 50

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CHAPTER 5 Experiments and results ........................................... 51

Validation of the PrepFiler™ Automated Forensic DNA Extraction Kit on the

HID EVOlution™—Extraction System ............................................ 52

Overview of experiments and results ......................................... 52

Materials and methods ..................................................... 52

Precision studies (SWGDAM standard 2.9) .................................... 54

Reproducibility studies (SWGDAM standard 2.5) ............................... 59

Correlation studies ......................................................... 61

Cross-contamination studies (SWGDAM standard 3.6) ......................... 64

STR studies ............................................................... 65

Veriﬁcation studies for remaining scripts ..................................... 69

Additional cross-contamination studies ...................................... 71

Conclusions .............................................................. 72

Validation of PrepFiler™ Wash Buer B and the related modiﬁcations to the

workstation layout and scripts ................................................. 73

Overview of experiments and results ......................................... 73

Materials and methods ..................................................... 74

Sample lysis method ....................................................... 77

Script validation ........................................................... 78

Precision and sensitivity studies ............................................. 80

Cross-contamination studies ................................................ 86

Case-type sample studies .................................................. 89

Comparative analysis studies ............................................... 93

Conclusions .............................................................. 99

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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APPENDIX A Troubleshooting .................................................. 100

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APPENDIX B Plate setup requirements for <96 samples .................... 104

Place samples in the plate (<96 samples) ........................................ 104

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APPENDIX C (One-time procedure) Document Te‑Shake™ plate

adapter temperatures ............................................................ 107

Required materials not supplied ................................................ 108

Measure the plate and adapter temperatures .................................... 108

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APPENDIX D Safety ............................................................. 111

Safety information for instruments not manufactured by Thermo Fisher Scientiﬁc ..... 112

Chemical safety .............................................................. 112

Biological hazard safety ....................................................... 114

Contents

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Documentation and support ...................................................... 115

Related documentation ........................................................ 115

Customer and technical support ................................................ 115

Limited product warranty ...................................................... 115

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Product description .................................................................... 6

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About the HID EVOlution™ systems ...................................................... 6

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Contents and storage .................................................................. 8

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Required materials not supplied ......................................................... 9

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IMPORTANT! Before using this product, read and understand the safety information in the

manufacturer's documentation.

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Product description

Product information

The PrepFiler™ Automated Forensic DNA Extraction Kit is part of an integrated solution that includes proven PrepFiler™ reagents for use in semi-automated, high-throughput workﬂows to provide reliable, simpliﬁed, and time-saving DNA extraction and puriﬁcation.

The kit uses magnetic particles with an optimized multi-component surface chemistry to deliver robust and reliable DNA yield from tested routine forensic sample types, including:

•

Body ﬂuids (blood, saliva, semen)

•

Stains and swabs of body ﬂuids

•

Hair roots

•

Touch/trace samples

About the HID EVOlution™ systems

The HID EVOlution™—Extraction System and HID EVOlution™—Combination System perform automated DNA puriﬁcation using a Freedom EVO™ robotic workstation with the PrepFiler™ Automated Forensic DNA Extraction Kit.

The robotic workstation automates liquid and magnetic particle handling, as shown in Figure 1. The puriﬁed DNA is collected in a 96-well plate or 1.5-mL tubes, depending on the Freedom EVOware software script that you select.

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PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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Chapter 1

About the HID EVOlution™ systems

Product information

Figure 1 Automated DNA puriﬁcation steps performed by the HID EVOlution™—Extraction System in plates (shown) or tubes (not shown)

Lysate is transferred from a spin plate or 1.5‑mL microfuge tubes into the PrepFiler™ 96-Well Processing Plate (shown) for automated DNA puriﬁcation.

Puriﬁcation

The automated puriﬁcation run time is ~3–4 hours for 96 samples, depending on the type of HID EVOlution™ system and conﬁguration that you are using.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

run times

Chapter 1 Product information

Contents and storage

System components

The HID EVOlution™—Extraction System and HID EVOlution™—Combination System consist of the following components.

•

A Freedom EVO™ 150 robotic workstation or Freedom EVO™ 200 robotic workstation

•

Freedom EVOware™ software v2.1 SP1 or later, conﬁgured with the HID EVOlution™—Extraction System application

Note: Contact Technical Support for more information on veriﬁed conﬁgurations. See “Customer and technical support” on page 115.

•

8-channel liquid-handling arm (LiHa)

•

Robotic Manipulator arm (RoMa)

•

Te‑Shake™ plate adapter with heating block and adapter

Plate/tube conﬁgurations

The HID EVOlution™ systems support the following plate/tube conﬁgurations. You can select one conﬁguration per puriﬁcation run.

•

Plate-to-plate—Process lysate from a 96-well plate and collect eluate in a 96-well plate

•

Plate-to-tubes—Process lysate from a 96-well plate and collect eluate in 1.5‑mL tubes

•

Tubes-to-tubes—Process lysate from 1.5‑mL tubes and collect eluate in 1.5‑mL tubes

•

Tubes-to-plate—Process lysate from 1.5‑mL tubes and collect eluate in a 96-well plate

Contents and storage

The PrepFiler™ Automated Forensic DNA Extraction Kit is intended for semi-automated workﬂows, and contains the reagents required for the following procedures:

•

Manual sample lysate preparation (DNA extraction)

•

Automated DNA puriﬁcation

The kit is sucient for ≤960 samples, depending on the batch size.

Table 1 PrepFiler™ Automated Forensic DNA Extraction Kit (Cat. No. 4463353)

Contents

PrepFiler™ Lysis Buer 1 × 500 mL 18–25°C

PrepFiler™ Magnetic Particles 13 × 1.5 mL

PrepFiler™ Wash Buer A Concentrate 1 × 500 mL

PrepFiler™ Wash Buer B Concentrate 1 × 250 mL

PrepFiler™ Elution Buer 1 × 200 mL

Amount Storage

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier. Catalog numbers that appear as links open the web pages for those products.

Table 2 Reagent preparation

Item Source

Chapter 1 Product information

Required materials not supplied

Ethanol (Molecular biology grade; 95% or 190 proof)

Note: Open a new bottle when preparing the PrepFiler™ Wash Buer A and Wash Buer B solutions.

Clean containers to store the prepared Wash Buer A and Wash Buer B solutions; we use:

Nalgene™ Square PETG Media Bottles with Closure: Sterile, ShrinkWrapped Trays

342020-0500 or 342020-1000

MLS

Table 3 Automated puriﬁcation

Item Source

Isopropanol (2‑Propanol, ACS reagent grade, ≥99.5% )

Note: Purchase isopropanol in small bottles and open fresh bottles frequently to maintain a high-quality grade reagent.

(If needed) DNA Suspension Buer (low-TE buer)

Note: DNA Suspension Buer is only needed if you run out of PrepFiler™ Elution Buer.

Magnetic-Ring Stand (96 well) AM10050

MLS

[1]

Disposable Tips (DiTi), Tecan™ Pure, Filtered, 1,000‑µL (30 000 631); six trays (each containing 96 DiTis)

Disposable Tips (DiTi), Tecan™ Pure, Filtered, 200‑µL (30 000 629); three trays (each containing 96 DiTis)

100‑mL disposable troughs for reagents (5 troughs) Tecan™ (10613048)

(Optional) Barcodes See the Tecan™ Freedom EVO

PrepFiler™ 96-Well Processing Plate (10 plates) A47010

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Tecan™ (30000631)

https://www.tecan.com/

Tecan™ (30000629)

https://www.tecan.com/

Operating Manual, Section 3.5.6

“Positive Identiﬁcation (PosID)”, for

barcode requirements

[2]

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Chapter 1 Product information

Required materials not supplied

Table 3 Automated puriﬁcation (continued)

[1]

If collecting eluate in plates:

Item Source

N8010560 or 4306737

MicroAmp™ Optical 96-Well Reaction Plate (without barcode) or MicroAmp™ Optical 96-Well Reaction Plate with Barcode

If collecting eluate in tubes:

AM12450, or equivalent

Nonstick, RNase-free Microfuge Tubes, 1.5 mL; certiﬁed DNase- and RNase-free (250 tubes)

[1]

Recommended sources. Unless otherwise indicated, equivalent materials from other suppliers can be used after appropriate validation studies by the user laboratory.

[2]

Disposable tips that have not been certified by Tecan™ may not yield the same liquid-handling performance.

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Prepare for the automated

puriﬁcation run

Perform lysis ......................................................................... 11

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Before ﬁrst use: Prepare the wash buers ............................................... 11

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Before each use: Prepare the magnetic particles ......................................... 11

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Perform lysis

Manually perform lysis according to the PrepFiler™ and PrepFiler™ BTA Automated Forensic DNA Extraction Kits User Guide (Pub. No. 4463349).

Before ﬁrst use: Prepare the wash buers

Mix 260 mL of PrepFiler™ Wash Buer A Concentrate with 740 mL of freshly-opened 95% ethanol in a separate, clean container to prepare a 1X solution.

Mix 200 mL of PrepFiler™ Wash Buer B Concentrate with 300 mL of freshly-opened 95% ethanol in a separate, clean container to prepare a 1X solution.

If the containers are kept closed when not in use, the prepared wash buers have a shelf life of 6 months or the kit expiration date, whichever is earlier.

Before each use: Prepare the magnetic particles

Incubate the PrepFiler™ Magnetic Particles tubes at 37℃ for 10 minutes.

Vortex at medium speed until the particles are completely resuspended and homogenous, then brieﬂy centrifuge.

Use one of the following methods to remove any air bubbles:

•

Draw o bubbles with a disposable bulb pipette.

•

Use a clean pipette tip to break up the bubbles.

•

Use a lint-free wipe to absorb the bubbles.

IMPORTANT! Bubbles can interfere with automated liquid detection and aspiration.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Set up the robotic workstation

For more information .................................................................. 12

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Workﬂow ............................................................................ 13

■

Perform routine maintenance .......................................................... 14

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(HID EVOlution™—Combination System only) Set up the carriers and labware ............... 16

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Set up the disposable pipette tips ...................................................... 19

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Set up reagents on the workstation ..................................................... 20

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Set up lysate, processing, and elution plates and/or tubes ................................. 23

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Workstation layouts ................................................................... 30

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For more information

This chapter provides general procedures for setting up the Freedom EVO™ 150 robotic workstation or Freedom EVO™ 200 robotic workstation for the HID EVOlution™ system.

For more information, see the appropriate manufacturer's documentation.

Document

Tecan™ HID EVOlution™—Extraction Application Manual

(395372, v2.0, June 2010)

Pre-run preparation step 4.3.2 “Prepare the Instrument”

Maintenance schedules 7.2, “Maintenance Schedule”

Maintenance procedures 7.3, “Maintenance Tasks”

Maintenance scripts 5.2, “Running Maintenance”

Setting up disposable pipette tips 4.3.5, “Setup Plasticware and

Setting up reagents 4.3.4, “Setup Reagents on

Place the plates and/or tubes 4.3.5, “Setup Plasticware and

Workstation layouts 4.3, "Preparing the Instrument"

Description Section

7.5, “Maintenance Scripts”

Samples on the Workstation”

Workstation”

Samples on the Workstation”

4.4, "Worktable Layouts"

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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(continued)

Chapter 3 Set up the robotic workstation

Document Description Section

Workﬂow

Tecan™ HID EVOlution™— Combination Application Manual

(395967, v2.0, June 2010)

Tecan™ Freedom EVO™ Operating Manual

Pre-run preparation step 5.3.2 “Prepare the Instrument”

Maintenance schedules 12.2, “Maintenance Schedule”

Maintenance procedures 12.3, “Maintenance Tasks”

Maintenance scripts 6.2 and 10.2, “Running Maintenance”

12.5, “Maintenance Scripts”

Setting up disposable pipette tips 5.3.5, “Set Up Plasticware and

Samples on the Workstation”

Setting up reagents 5.3.4, “Set Up Reagents on

Workstation”

Place the plates and/or tubes 5.3.5, “Set Up Plasticware and

Samples on the Workstation”

Workstation layouts 5.3, "Preparing the Instrument"

5.4, "Worktable Layouts"

Barcode speciﬁcations 3.5.6, “Positive Identiﬁcation (PosID)”

Workﬂow

Prepare the reagents and samples

Prepare the system liquid carboy

(HID EVOlution™—Combination System only) Set up the carriers and labware

Set up the disposable pipette tips

Set up reagents on the workstation

Set up lysate, processing, and elution plates and/or tubes

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3 Set up the robotic workstation

Perform routine maintenance

Before placing the samples, reagents, and labware (DiTis, troughs, plates, and tubes), prepare the robotic workstation.

Prepare the system liquid carboy

Ensure that the carboy next to the workstation contains enough system liquid (degassed deionized water) to complete the experiment.

Degas the deionized water overnight or longer before using it on the system.

Note: The time needed for complete degassing varies, depending on the climate in each laboratory and geographical location. In some situations, it may take up to 3 days to fully degas the deionized water. We recommend that each laboratory maintain an additional carboy of fully degassed deionized water to use for replenishment.

To avoid introducing air into the system liquid tubing, follow these guidelines:

•

Place the system liquid carboy at the same height as the worktable.

•

Replenish the system liquid as needed before each run to avoid liquid levels dropping below one-quarter carboy during the run.

Run the routine maintenance script each time that you change the system liquid carboy.

Empty the waste carboy

Check the waste carboy, and empty if needed.

Tighten the DiTi adapter gold cones

Note: If the cones are loose, the instrument may fail to pick up pipette tips during the run, and liquid

delivery will be inconsistent.

Use your ﬁngers to gently tighten the DiTi adapter gold cones on the LiHa and the syringe assembly

ﬁttings.

For details, see the Tecan™ Freedom EVO™ Operating Manual.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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Run maintenance scripts

Before starting the run, run the appropriate maintenance scripts.

IMPORTANT! Watch for air bubbles in the syringes and tubing. Repeat system ﬂushing as needed to

remove the air bubbles.

If Then run

It is the ﬁrst run of the day PrepFiler_DailyStartUp or Combo_DailyStartUp

It is not the ﬁrst run of the day PrepFiler_Flush or Combo_Flush

Chapter 3 Set up the robotic workstation

Perform routine maintenance

When you run DailyStartUp or Flush, you see:

•

Air bubbles in the lines

and/or

•

Intermittent ﬂow from a DiTi cone

There are one or more DiTis on the LiHa PrepFiler_Drop_DiTis or Combo_Drop_DiTis

PrepFiler_Flush or Combo_Flush one or more times until:

•

There are no visible air bubbles

and

•

Flow from the DiTi cones is constant

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3 Set up the robotic workstation

(HID EVOlution™—Combination System only) Set up the carriers and labware

If the HID EVOlution™—Combination System was last run for qPCR/STR, you will need to set up the carriers and labware for a puriﬁcation run.

If the DNA lysate is in tubes, remove the 3-position microplate carrier from Grid 7, then place six tube racks on grids 7 through 12.

Note: If the DNA lysate is in a plate, you do not need to remove the 3-position microplate carrier.

•

3-position microplate carrier

Set up carriers for the DNA eluate.

•

If you want the DNA eluate placed in tubes, position six tube racks on grids 1 through 6.

•

If you want the DNA eluate placed in a 96-well plate, position the metal plate adaptor on grid 13, position 1.

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Chapter 3

(HID EVOlution™—Combination System only) Set up the carriers and labware

Remove the 3-position disposable tips (DiTi) tray carrier from grid 35, then replace it with a ﬂat

Set up the robotic workstation

carrier and three 1,000-μL DiTi boxes as shown.

•

200-µL DiTi trays (blue or white trays)

•

1,000-µL DiTi trays (yellow or white trays)

Place the magnetic particle tube block on grid 13, position 2.

IMPORTANT! Ensure that the tubes and the block containing the tubes are positioned as shown.

Incorrect positioning may result in failure to pipet magnetic particles and/or collision of the Liquid Handling (LiHa) arm with the block.

Ensure that the 96-well magnetic ring stand is on grid 19, position 2.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3

(HID EVOlution™—Combination System only) Set up the carriers and labware

Set up the robotic workstation

Set up the reagent troughs.

Remove the reagent troughs from previous runs and correctly dispose of the reagents according to “Complete the run” on page 48.

Place new 100-mL reagent troughs on the worktable for PrepFiler™ Wash Buer B and other reagents as shown.

Note: The trough layout shown is dierent from the originally validated layout. The validation of the new trough layout is described in “Validation of PrepFiler™ Wash Buer B and the related modiﬁcations to the workstation layout and scripts” on page 73.

Elution buer trough (grid 27, position 1)

Prepared Wash Buer B trough (grid 27, position 2)

Prepared Wash Buer A trough (grid 27, position 3)

Isopropanol trough (grid 25, position 1)

Lysate waste trough (grid 25, position 3)

The workstation should now match the setup shown in“Workstation layouts” on page 30.

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Chapter 3 Set up the robotic workstation

Set up the disposable pipette tips

Terms for pipette tips used on the robotic workstation

•

DiTis—Disposable Tips (DiTi), Tecan™ Pure, Filtered, 200- and 1,000‑µL

•

DiTi tray—Plastic tray containing 96 DiTis

•

DiTi rack—Aluminum holder for a single tray of 1,000‑µL DiTis

•

DiTi carrier—Aluminum holder for three trays of 200‑µL DiTis

•

Orientation nose—Pin on a DiTi rack to hold the tray in place

Set up the disposable pipette tips

Figure 2 DiTi terms.

Racks that contain 1,000-μL DiTis on the rear shelf positions 5, 6, and 7

Notch in the DiTi tray

Carrier for 200-μL DiTi trays (trays are blue or white)

Orientation nose

Racks that contain 1,000-μL DiTi trays (trays are yellow or white)

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3 Set up the robotic workstation

Set up reagents on the workstation

Fill DiTi carriers and racks

Set up the pipette tips for an automated puriﬁcation run.

IMPORTANT! If nine full DiTi trays are not correctly set up on the Freedom EVO

the workstation repeatedly searches for the missing DiTi tips, during which time the samples may become unusable.

Place three full trays of 1,000‑µL DiTis into the DiTi racks on the rear shelf (shelf positions 5, 6, and

7). For each tray:

Insert the tray into a rack: Ensure that the notch in the tray is aligned with the orientation nose on the rack, snap the tray into the rack, then conﬁrm that the tray ﬁts snugly.

Place the rack on the shelf: Ensure that the orientation pin is positioned toward the back of the shelf, then push the rack all the way to the back of the shelf.

™

robotic workstation,

IMPORTANT! Ensure that there are no objects placed on shelf positions 1–4 or position 8.

Place three full trays of 1,000‑µL DiTis into the DiTi racks on grid 35, positions 1–3, as described in substep 1a. Ensure that the orientation pin is positioned in the top-left corner.

Place three full trays of 200‑µL DiTis into the carrier on grid 29, positions 1–3. Ensure that the notch in the tray is positioned in the top-left corner.

IMPORTANT! Ensure that the 3-position DiTi carrier on grid 29 contains three 200-μL DiTi trays.

Set up reagents on the workstation

Procedural guidelines

•

Calculate the reagent volumes needed based on the number of samples you will process plus the speciﬁed overﬁll and dead volumes.

Note: The dead volume is independent of the number of samples you run.

•

Do not reuse isopropanol, PrepFiler™ Wash Buer A, PrepFiler™ Wash Buer B, or PrepFiler Elution Buer from previous runs; always properly dispose of used reagents after each run.

•

Do not use water instead of PrepFiler™ Elution Buer. Instead of PrepFiler™ Elution Buer, you can prepare low-TE buer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) or purchase DNA Suspension Buer (low-TE Buer) from Teknova™.

•

Use new reagent troughs each day.

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PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User

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Set up the reagents

Place empty troughs on the workstation according to the following table.

PrepFiler™ Elution Buer Grid 27, position 1

PrepFiler™ Wash Buer B Grid 27, position 2

PrepFiler™ Wash Buer A Grid 27, position 3

Isopropanol Grid 25, position 1

Chapter 3 Set up the robotic workstation

Set up reagents on the workstation

Empty trough Location

Lysate waste

IMPORTANT! Do not add acids or bases to

any wastes that contain PrepFiler™ Lysis Buer (guanidine thiocyanate).

Calculate the required PrepFiler™ reagent volumes.

Reagent

Lysis

protocol

Isopropanol Standard,

300‑µL

Largesample,

[4]

Prepared Wash

500‑µL

— Up to 96 900 µL 15% 5 mL 105 mL

Buer A

Prepared Wash

— Up to 96 300 µL 15% 5 mL 40 mL

Buer B

Grid 25, position 3

No. of

reactions

Reagent

volume per

reaction

Overﬁll

volume per

[1]

run

Dead

volume per

[2]

run

Minimum required

volume for

96 samples

(A×B)+(A×B×C)

A B C D

Up to 96 180 µL 15% 5 mL 25 mL

Up to 96 300 µL 15% 5 mL 40 mL

[3]

Elution Buer — Up to 96 50 µL 15% 5 mL 11 mL

[1]

Overfill (excess volume) is needed to compensate for evaporation and pipetting losses during the run.

[2]

An extra 5 mL per trough is needed to ensure that the pipette tips remain submerged during aspiration so that liquid, not air, enters the tips.

[3]

Includes overfill and dead volume. For example, the required volume of isopropanol for 96 samples when using the standard lysis protocol is (96 × 180 µL) + (96 × 180 µL × 0.15) + 5 mL = 17.28 mL + 2.59 mL + 5 mL = 24.87 mL, rounded up to 25 mL.

[4]

The large-sample (500‑µL) protocols were not tested as part of our full validation studies. If your laboratory intends to use the large-sample protocols, perform the appropriate validation studies.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3

Set up reagents on the workstation

Set up the robotic workstation

Add the amounts of PrepFiler™ reagents that you calculated in step 2 to the appropriate trough.

Elution buer trough

Prepared Wash Buer B trough

Prepared Wash Buer A trough

Isopropanol trough

Lysate waste trough, empty

Gently invert two tubes of prepared PrepFiler™ Magnetic Particles to remove large air bubbles, brieﬂy centrifuge the tubes at low speed to collect the contents at the bottom of the tubes, then open the tubes.

•

If a thin ﬁlm or bubble (caused by surfactants) stretches across the top of the tube, gently break the surface with a clean pipette tip.

•

If there is foam (air bubbles) on the surface of the magnetic particles, remove the foam by pipetting. Surface foam may interfere with liquid level detection during the automated puriﬁcation run.

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Chapter 3

Set up lysate, processing, and elution plates and/or tubes

Place the two tubes of PrepFiler™ Magnetic Particles on the workstation in the ﬁrst two slots of the

Set up the robotic workstation

metal rack on grid 13, position 2.

IMPORTANT! Ensure that the tubes and the block containing the tubes are positioned as shown.

Incorrect positioning may result in failure to pipette magnetic particles and/or collision of the Liquid Handling (LiHa) arm with the block.

Set up lysate, processing, and elution plates and/or tubes

Select a plate/tube conﬁguration

The HID EVOlution™ systems support four plate/tube conﬁgurations for the Freedom EVO™ 150 robotic workstation or Freedom EVO™ 200 robotic workstation.

Select a conﬁguration for each automated puriﬁcation run.

Starting labware: Plate or tube

Conﬁguration

Plate-to-plate Process lysate from a 96-well

plate and collect eluate in a 96well plate

Plate-to-tubes Process lysate from a 96-well

plate and collect eluate in

1.5‑mL tubes

Tubes-to-tubes Process lysate from 1.5‑mL

tubes and collect eluate in

1.5‑mL tubes

Tubes-to-plate Process lysate from 1.5‑mL

tubes and collect eluate in a 96well plate

[1]

Your choice is independent of whether the sample lysate is contained in a plate or in tubes

Description

that contains the lysate from

the sample lysis step

PrepFiler™ Spin Plate MicroAmp™ Optical 96-Well

PrepFiler™ Spin Plate Nonstick RNase-free

Nonstick RNase-free Microfuge Tubes (1.5‑mL)

Ending labware: Plate or tube

to collect DNA eluate at the

end of the run

Reaction Plate

Microfuge Tubes (1.5‑mL)

Nonstick RNase-free Microfuge Tubes (1.5‑mL)

MicroAmp™ Optical 96-Well Reaction Plate

[1]

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3

Set up lysate, processing, and elution plates and/or tubes

“Workstation layouts” on page 30 shows the placement of plates and tubes for each conﬁguration.

Set up the robotic workstation

Set up the PrepFiler™ Processing Plate

The PrepFiler™ Processing Plate is a square-well plate that is required to process reactions for all four automated puriﬁcation run conﬁgurations.

During the washing and elution steps, the Robotic Manipulator arm (RoMa) moves the processing plate to the 96-Well Magnetic Ring Stand or Te‑Shake™ plate adapter.

If needed to ensure that the RoMa grips the plate tightly, place a strip of laboratory labeling tape on each side of the PrepFiler™ Processing Plate as shown.

Place the PrepFiler™ Processing Plate on the Te‑Shake™ plate adapter with well A1 in the top-left position (grid 19, position 3).

To ensure that samples are transferred to the correct wells, conﬁrm that:

•

The processing plate is placed on the Te‑Shake™ plate adapter with well A1 positioned in the top-left corner

•

The plate wells are aligned with the holes in the Te‑Shake™ plate adapter

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(If needed) Place barcodes

Perform this procedure if you use barcodes on the plates and/or tubes to track the samples in the HID EVOlution™ software. The system scans the barcodes to automatically capture sample information. For more information, see the Tecan™ HID EVOlution™ —Extraction Application Manual, Section 4.5, “Barcodes”.

IMPORTANT!

If the lysate is in spin/ﬁlter plates: Before the plate barcode is scanned during a run, you must

manually enter or import the sample information for each well in the plate. (See “Set up sample and reagent information” on page 40, step 1.) If the lysate is in tubes: The sample name (barcode) and sample position for each tube are

automatically updated in the HID EVOlution™ software when the barcodes are scanned.

Select barcodes that are compatible with the PosID-3.

Before placing items on the robotic workstation, ensure that the barcodes are correctly placed on the appropriate labware.

Chapter 3 Set up the robotic workstation

Set up lysate, processing, and elution plates and/or tubes

Component

Sample lysate PrepFiler™ Spin Plates Nonstick RNase-free Microfuge

DNA eluate MicroAmp™ Optical 96-Well Reaction

Plate

Set up lysate and/or eluate plates

IMPORTANT! To ensure that samples are transferred to the correct wells, conﬁrm the following for

each lysate or eluate plate:

The plate is placed in the metal plate adapter with well A1 positioned in the upper left corner

The plate wells are aligned with the holes in the metal plate adapter

Plate Tube

Tubes (1.5‑mL)

Nonstick RNase-free Microfuge Tubes (1.5‑mL)

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3 Set up the robotic workstation

Set up lysate, processing, and elution plates and/or tubes

If you want DNA eluate to be collected in a plate, place a MicroAmp™ Optical 96-Well Reaction Plate with well A1 in the top-left position (grid 13, position 1).

Note: The DNA eluate corresponding to the ﬁrst sample is always placed in the ﬁrst well (A1) of the elution plate. The Report ﬁle (PDF) that is generated at the end of the puriﬁcation run lists the starting position of each sample lysate and the ﬁnal position of the corresponding DNA eluate.

Note: Using 96-well plates from other manufacturers may result in liquid handling errors if the instrument is not recalibrated for use with the alternate plates.

If the lysate is in a PrepFiler™ Spin Plate, place the spin plate with well A1 in the top-left position (grid 13, position 3).

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Set up lysate and/or eluate tubes

Set up eluate tubes in tube racks

Ensure that:

•

You have new, labeled 1.5‑mL microfuge tubes equal to the number of DNA samples to be processed.

•

The tube racks S1–S6 are correctly positioned at grid positions 1–6.

Place the ﬁrst empty 1.5‑mL microfuge tube in the tube racks in rack S1, position 1.

Note: The DNA eluate corresponding to the ﬁrst sample is always placed in the ﬁrst tube (1) in the ﬁrst tube rack (S1). The Report ﬁle (PDF) that is generated at the end of the puriﬁcation run lists the starting position of each sample lysate and the ﬁnal position of the corresponding DNA eluate.

Continue placing empty tubes from back to front in vertical columns as shown. Place one empty tube for each sample to be processed. Do not leave empty positions between sample tubes.

IMPORTANT! The tubes must be contiguously loaded. Do not leave empty tube positions

between tubes.

Chapter 3 Set up the robotic workstation

Set up lysate, processing, and elution plates and/or tubes

Example of correct setup

Ensure that the barcodes are in a readable position.

Open each tube, securing the tube caps in a ﬁxed upright position as shown.

IMPORTANT! Open tube caps carefully to prevent cross-contamination and splatter.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3

Set up lysate, processing, and elution plates and/or tubes

Set up the robotic workstation

Set up lysate tubes in tube racks

Ensure that:

•

You have ≤96 labeled 1.5‑mL microfuge tubes that contain DNA sample lysate.

•

The tube racks L1–L6 are correctly positioned at grid positions 7–12.

Place the ﬁrst sample tube in the tube racks. (Unlike the ﬁrst eluate tube, which must be placed in rack S1, position 1, the ﬁrst lysate tube may be placed in any position; for example, you can begin with rack L1, position 8.)

Continue placing sample tubes from back to front in vertical columns as shown. Do not leave empty positions between sample tubes.

IMPORTANT! The tubes must be contiguously loaded. Do not leave empty tube positions

between tubes.

Examples of correct setup

Ensure that the barcodes are in a readable position.

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Chapter 3 Set up the robotic workstation

Set up lysate, processing, and elution plates and/or tubes

Open each tube, securing the tube caps in a ﬁxed upright position as shown.

IMPORTANT! Open tube caps carefully to prevent cross-contamination and splatter.

PrepFiler™ Automated Forensic DNA Extraction Kit: Automated DNA Puriﬁcation on the HID EVOlution™ Systems User Bulletin

Chapter 3 Set up the robotic workstation

Workstation layouts

The following ﬁgures show the available workstation layouts for the automated puriﬁcation run conﬁgurations.

Figure 3 Plate-to-plate workstation layout

96-Well Elution Plate

Block for PrepFiler™ Magnetic Particles

PrepFiler™ Spin Plate

Magnetic Ring Stand

PrepFiler™ Processing Plate on Te‑Shake™ plate adapter

Isopropanol trough

Lysate waste trough

Elution Buer trough

Wash Buer B trough

Wash Buer A trough

DiTi waste unit

200‑µL disposable pipette tips (DiTis)

1,000‑µL DiTis

Not shown: Rear shelf with 1,000‑µL DiTis in shelf positions 5, 6, and 7

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