Chargeswitch GDNA - Buccal - Manual

8/11/2019 Chargeswitch GDNA - Buccal - Manual

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Instruction Manual

ChargeSwitchgDNABuccal Cell

Kits

Forpurification of genomic DNA fromhuman

Catalog nos. CS11020, CS11021, CS11020-10,andCS11021-10

buccal swabs

Version A

7Janua ry 2005

25-0819


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ii


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Table of Contents

Table of Contents..................................................................................................iii

Kit Contents and Storage ..................................................................................... v

Accessory Products..............................................................................................vi

Introduction ........................................................................................1

Overview .................................................................................................................1

Experimental Outline ............................................................................................4

Methods............................................................................................... 5

General Information Individual Samples .......................................................5

Isolating Genomic DNA from Individual Samples ..........................................8

General Information Automated Sample Processing .................................14

Automated Genomic DNA Isolation ................................................................18

Automated Genomic DNA Isolation Normalized Kit ................................23

Troubleshooting ...................................................................................................27

Appendix...........................................................................................30

Technical Service ..................................................................................................30

Purchaser Notification and Product Qualification .........................................32

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Kit Contents and Storage

Types of Kits This manual is supplied with the following products.

Product Number ofPurifications

Catalog no.

ChargeSwitchgDNA Normalized Buccal CellKit

50

960

CS11020

CS11020-10

ChargeSwitchgDNA Buccal Cell Kit 50

960

CS11021

CS11021-10

Shipping andStorage

All components of the ChargeSwitchgDNA Buccal CellKits are shipped at room temperature. Upon receipt, store

the Proteinase K at 4C. Store all other components at roomtemperature.

All components are guaranteed stable for 6 months, ifstored properly.

Contents The components supplied in the ChargeSwitchgDNABuccal Cell Kits are listed below.

Note:Some reagents in the kit may be provided in excess of theamount needed.

Catalog no.

Components CS11020 CS11021 CS11020-10 CS11021-10

ChargeSwitch

Lysis Buffer(L11) 50 ml 50 ml 960 ml 960 ml

ChargeSwitchMagneticBeads

2 x 1 ml 2 x 1 ml 40 ml 40 ml

Proteinase K (20 mg/ml in 50mM Tris-HCl, pH 8.5, 5 mMCaCl2, 50% glycerol)

500 l 500 l 9.6 ml 9.6 ml

ChargeSwitchPurification

Buffer (N6)

5 ml 5 ml 100 ml 125 ml

ChargeSwitchWash Buffer(W12)

100 ml 100 ml 2 x 960 ml 2 x 960 ml

ChargeSwitchElution Buffer(E5; 10 mM Tris-HCl, pH 8.5)

15 ml 15 ml 2 x 100 ml 2 x 100 ml

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Accessory Products

Additional

Products

The table below lists additional products available from

Invitrogen that may be used with the ChargeSwitchgDNABuccal Cell Kits. In addition, the table lists a selection ofChargeSwitchgDNA Kits that are available for purificationof genomic DNA from other sources. For more informationabout these and other ChargeSwitchgDNA Kits, refer toour Web site at www.invitrogen.comor call TechnicalService (see page 30).

Product Amount Catalog no.

MagnaRack 1 rack CS15000

96-Well Magnetic Separator 1 rack CS15096

ChargeSwitchgDNA Micro Tissue Kit 50 purifications CS11203

ChargeSwitchgDNA Mini Tissue Kit 25 purifications CS11204

ChargeSwitchgDNA 20 l Blood Kit 96 purifications CS11010

ChargeSwitchgDNA 100 l Blood Kit 50 purifications CS11000

ChargeSwitchgDNA 1 ml Blood Kit 20 purifications CS11001

ChargeSwitchgDNA 1 ml Serum Kit 50 purifications CS11040

ChargeSwitchgDNA 50 l Sheep BloodKit

50 purifications CS11300

ChargeSwitchgDNA Mini Bacteria Kit 50 purifications CS11301

ChargeSwitchForensic DNAPurification Kit

100 purifications CS11200

Quant-iTDNA Assay Kit, HighSensitivity

1000 assays Q33120

vi
http://www.invitrogen.com/http://www.invitrogen.com/


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Introduction

Overview

Introduction The ChargeSwitchgDNA Buccal Cell Kits allow rapid andefficient purification of genomic DNA from human buccalswabs. After preparing the lysates, you may purify DNA inless than 15 minutes using the ChargeSwitchTechnology.Depending on the kit used, samples may be handledindividually or in an automated system using a liquidhandling robot. For more information about the

ChargeSwitch

Technology, see page 3.

Intended Usefor the Kits

The ChargeSwitchgDNA Buccal Cell Kits are designed toallow isolation of the following amounts of genomic DNAfrom human buccal cell swabs or pelleted cells from a

mouthwash. Samples can be stored for up to 2 weeks at 4Cbefore processing without a noticeable loss in DNA yield orquality. The purified genomic DNA is suitable for use in

downstream applications such as PCR.

ChargeSwitchgDNA Normalized Buccal Cell Kits:Produce a normalized yield of genomic DNA at a

concentration of 1-3 ng/l in a total volume of 150 l.

ChargeSwitchgDNA Buccal Cell Kits:Purify up to

6 g of genomic DNA.

Important:The DNA yield varies and is dependent on several factorsincluding the technique of the person taking the swab, whether the

donor is a high or low shedder, and the type of swab used.

continued on next page

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Overview, continued

Advantages Use of the ChargeSwitchgDNA Buccal Cell Kits to isolate

genomic DNA provides the following advantages:

Uses a magnetic bead-based technology to isolategenomic DNA without the need for hazardouschemicals, centrifugation, or vacuum manifolds

Rapid and efficient purification of genomic DNA fromhuman buccal swabs in less than 15 minutes followingsample preparation and lysis

Simple lysis with Proteinase K without the need for anymechanical lysis

Minimal contamination with RNA

The purified genomic DNA demonstrates improveddownstream performance in applications including PCR

Includes a kit designed for automated processing of largenumbers of samples in 96-well plates using a liquidhandling robot

SystemSpecifications

Starting Material: Human buccal swabs

Elution Volume: 150 l

DNA Yield: 1-3 ng/l in 150 l (Normalized Buccal

Cell Kit) or up to 6 g (Buccal Cell Kit)

DNA Size: Varies (depends on quality of startingmaterial


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Overview, continued

The

ChargeSwitchTechnology

The ChargeSwitchTechnology (CST) is a novel magnetic

bead-based technology that provides a switchable surfacecharge dependent on the pH of the surrounding buffer tofacilitate nucleic acid purification. In low pH conditions, theCSTbeads have a positive charge that binds the negativelycharged nucleic acid backbone (see figure below). Proteinsand other contaminants are not bound and are simplywashed away in an aqueous wash buffer. To elute nucleicacids, the charge on the surface of the bead is neutralized byraising the pH to 8.5 using a low salt elution buffer (see

figure below). Purified DNA elutes instantly into this elutionbuffer, and is ready for use in downstream applications.

ChargeSwitch

Magnetic BeadSpecifications

Bead Binding Capacity: 5-10 g genomic DNA per mg

Bead Size: < 1 m

Bead Concentration: 25 mg/ml (Buccal Cell Kitsonly)

3.125 mg/ml (NormalizedBuccal Cell Kits only)

Storage Buffer: 10 mM MES, pH 5.0, 10 mMNaCl, 0.1% Tween 20

AutomatedLiquidHandling

Use of the ChargeSwitchgDNA Buccal Cell Kits has beendemonstrated on the Tecan Genesisrobotic workstation topurify DNA in a fully automated system from largenumbers of buccal cell swabs in a 96-well format. Otherliquid handling robots are suitable provided that each isequipped with a gripper arm, a 96-well magnetic separator,and other additional hardware as described on page 14. Thismanual provides general guidelines and a protocol that may

be used to develop a script for your robot. For moreinformation, see www.invitrogen.comor call TechnicalService (page 30).

Genesisis a registered trademark of Tecan AG Group

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Experimental Outline

Introduction The figure below illustrates the basic steps necessary to

purify genomic DNA from your buccal cell swab using oneof the ChargeSwitchgDNA Buccal Cell Kits.

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Methods

General Information Individual Samples

Introduction This section provides general information needed to use theChargeSwitchgDNA Buccal Cell Kits (Catalog nos.CS11020 or CS11021) to process individual samples. If youare using a liquid handling robot to process large numbersof samples, see General Information Automated SampleProcessing, page 14.

User SuppliedMaterials

In addition to the reagents supplied with the kit, you need tohave the following materials on hand before beginning:

A magnetic separation rack suitable for use with 1.5 mlmicrocentrifuge tubes (see below)

Sterile, 1.5 ml microcentrifuge tubes

Vortex mixer

20 l, 200 l, and 1 ml sterile, pipette tips

Water bath at 37C

MagnaRack

The MagnaRackavailable from Invitrogen (Catalog no.CS15000) is a two-piece magnetic separation rack for use inprotocols with magnetic beads, and consists of a magnetic

base station and a removable tube rack. The tube rack canhold up to 24 microcentrifuge tubes. The tube rack fits ontothe magnetic base station in two different positions,

associating the row of 12 neodymium magnets with a singlerow of 12 tubes for simple on the magnet and off themagnet sample processing (see figure below). For moreinformation, see www.invitrogen.comor call TechnicalService (page 30).


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General Information Individual Samples,continued

SafetyInformation

Follow the safety guidelines below when using theChargeSwitchgDNA Buccal Cell Kits.

Treat all reagents supplied in the kit as potentialirritants.

Always wear a suitable lab coat, disposable gloves, andprotective goggles.

If a spill of the buffers occurs, clean with a suitable

laboratory detergent and water. If the liquid spillcontains potentially infectious agents, clean the affectedarea first with laboratory detergent and water, thenwith 1% (v/v) sodium hypochlorite or a suitablelaboratory disinfectant.

Dispose of biological samples and all liquid wastegenerated during the purification procedure as

biohazardous waste.

Handling theChargeSwitch

MagneticBeads

Follow the guidelines below when handling theChargeSwitchmagnetic beads.

Do not freeze the beads as this irreparably damagesthem. Store the beads at room temperature.

Always keep the beads in solution. Do not allow themto dry out as this renders them non-functional.

When using the beads, resuspend thoroughly in thestorage buffer by vortexing before removal.

Discard beads after use. Do not reuse.


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General Information Individual Samples,continued

Elution Buffer ChargeSwitchElution Buffer (E5; 10 mM Tris-HCl, pH 8.5)is supplied with the kit for eluting the DNA from theChargeSwitchMagnetic Beads. For best results, use ElutionBuffer (E5) to elute the DNA. Alternatively, TE Buffer, pH8.5-9.0 is acceptable. Note that the pH must be between 8.5-9.0 otherwise the DNA will not elute. Do not use water forelution.

The protocol suggests eluting the genomic DNA in 150 l of

ChargeSwitchElution Buffer (E5). When using theChargeSwitchgDNA Buccal Cell Kit, you may vary theamount of ChargeSwitchElution Buffer (E5) used to obtaingenomic DNA in the desired final concentration. For bestresults, always use a volume of ChargeSwitchElutionBuffer (E5) that is equal to or greater than the volume ofChargeSwitchMagnetic Beads used in the protocol.If thevolume of ChargeSwitchElution Buffer (E5) is lower thanthe volume of beads used, DNA elution is incomplete. You

may need to perform a second elution to recover all DNA.Important:When using the ChargeSwitchgDNA Normalized

Buccal Cell Kit, elute the genomic DNA in 150 l of ChargeSwitchElution Buffer (E5). Do notvary the amount of ChargeSwitchElution Buffer (E5) used otherwise the yield will no longer be

normalized at 1-3 ng/l.

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Isolating Genomic DNA from IndividualSamples

Introduction This section provides guidelines and instructions to isolategenomic DNA from human buccal swabs or pelleted cellsfrom a mouthwash using the reagents supplied in the kit.Note that the protocol is optimized for efficient purificationof DNA from small sample volumes. Depending on thevolume of your sample, some further optimization of theprotocol may be required.

StartingMaterial

Use this procedure to isolate genomic DNA from humanbuccal cell swabs or pelleted cells from a mouthwash.

Process samples immediately after collection or store at 4Cfor up to 2 weeks. Do notstore unprocessed samples atroom temperature as buccal swabs contain bacteria andnucleases that will degrade DNA.

The ChargeSwitchMagnetic Beads supplied in theChargeSwitchgDNA Buccal Cell Kit differ inconcentration from those supplied in the ChargeSwitchgDNA Normalized Buccal Cell Kit.Although thepurification protocol is identical for both kits, you mustusethe ChargeSwitchMagnetic Beads supplied with each kit toobtain the DNA yields specified on page 1. Do notsubstitute ChargeSwitchMagnetic Beads provided in theChargeSwitchgDNA Buccal Cell Kit for those in the

ChargeSwitch

gDNA Normalized Buccal Cell Kit.


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Isolating Genomic DNA from IndividualSamples, continued

MaterialsNeeded

Have the following materials on hand before beginning:

Buccal swab(s)

MagnaRack(Catalog no. CS15000)

Sterile 1.5 ml microcentrifuge tubes

Vortex mixer

Sterile pipette tips (20 l, 200 l, and 1 ml)

Water bath

Components Supplied with the Kit

ChargeSwitchLysis Buffer (L11)

Proteinase K

ChargeSwitchMagnetic Beads

ChargeSwitchPurification Buffer (N6)

ChargeSwitch

Wash Buffer (W12) ChargeSwitchElution Buffer (E5) or TE Buffer (not

supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)


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BeforeStarting

Perform the following before beginning:

1. Set a water bath at 37C.

2. Prepare a Lysis Mix: For each sample, mix 1 ml of

ChargeSwitchLysis Buffer (L11) and 10 l of ProteinaseK to prepare the Lysis Mix. If you are isolating DNAfrom multiple samples, you may scale up the volume ofreagents used and prepare a master Lysis Mix.

3. Vortex the tube containing the ChargeSwitchMagneticBeads to fully resuspend and evenly distribute the beadsin the storage buffer.

4. Prepare a Purification Mix: For each sample, mix 40 lof ChargeSwitchMagnetic Beads (fully resuspended;

see above) and 100 l of ChargeSwitchPurificationBuffer (N6) to prepare the Purification Mix. If you areisolating DNA from multiple samples, you may scale upthe volume of reagents used and prepare a masterPurification Mix.

Preparing theLysate

Follow the procedure below to prepare a lysate from thehuman buccal cell swab.

1. Transfer the human buccal cell sample to a sterilemicrocentrifuge tube.

2. Add 1 ml of Lysis Mix (see above) to the tube, making

sure that the sample is completely immersed in the LysisMix.

3. Incubate the sample at 37C for 20 minutes.

4. Proceed to Binding DNA, next page.


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Binding DNA Follow the procedure below to bind the DNA to theChargeSwitchMagnetic Beads.

1. Transfer the digested supernatant (from Step 3, previouspage) into a new, sterile microcentrifuge tube.

2. Gently pipet up and down the Purification Mixcontaining the ChargeSwitchMagnetic Beads (seeprevious page) to fully resuspend the beads.

3. Add 140 l of Purification Mix to the sample and pipetup and down gently 5 times to mix.

Important:Use a 1 ml pipette tip set to 900 l to mix the sample.Make sure that the tip is submerged, and pipet up and downgently to avoid forming bubbles.

4. Incubate at room temperature for 1 minute to allow theDNA to bind to the ChargeSwitchMagnetic Beads.

5. Place the sample in the MagnaRackfor 1 minute or until

the beads have formed a tight pellet.6. Without removing the tube from the MagnaRack,

carefully remove the supernatant and discard. Take carenot to disturb the pellet of beads by angling the pipettesuch that the tip is pointed away from the pellet (seefigure below).

7. Proceed immediately to Washing DNA, next page.


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Washing DNA 1. Without removing the tube from the MagnaRack, add1 ml of ChargeSwitchWash Buffer (W12) to the sample.Direct the ChargeSwitchWash Buffer over the pellet ofmagnetic beads in such a way that the beads are brieflyresuspended in solution.

2. Leave the sample in the MagnaRackfor 1 minute oruntil the beads have formed a tight pellet.

3. Without removing the tube from the MagnaRack,carefully remove the supernatant and discard. Take carenot to disturb the pellet of beads by angling the pipettesuch that the tip is pointed away from the pellet (seefigure on page 11).

4. Repeat Steps 1-3.

5. Proceed to Eluting DNA, below.

Eluting DNA 1. Remove the tube containing the pelleted magnetic beadsfrom the MagnaRack(Step 4, above). There should beno supernatant in the tube.

2. Add 150 l of ChargeSwitchElution Buffer (E5) (or TEBuffer, pH 8.5) to the tube and pipet up and down gently10 times to resuspend the magnetic beads.

Important: Do not use water for elution. The DNA will not elutedue to the poor buffering capacity of water.

3. Incubate at room temperature for 1 minute.

4. Place the sample in the MagnaRackfor 1 minute or untilthe beads have formed a tight pellet.

5. Without removing the tube from the MagnaRack,carefully remove the supernatantcontaining the DNAtoa sterile microcentrifuge tube. Take care not to disturbthe pellet of beads by angling the pipette such that the tipis pointed away from the pellet (see figure on page 11).

Note:If the eluate containing the DNA is discolored, repeatSteps 4-5.

6. Discard the used magnetic beads. Do not reuse the beads.


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Storing DNA Store the purified DNA at -20C or use immediately for PCRor other appropriate downstream application. Avoidrepeatedly freezing and thawing DNA.

QuantitatingDNA Yield

To quantitate yield of your DNA, we recommend using theQuant-iTDNA Assay Kit, High Sensitivity (Catalog no.Q33120) available from Invitrogen. This kit contains a state-

of-the-art quantitation reagent, pre-diluted standards, and apre-made buffer to allow sensitive and accuratefluorescence-based quantitation of dsDNA. For moreinformation about the Quant-iTDNA Assay Kit, seewww.invitrogen.comor call Technical Service (page 30).

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General Information Automated SampleProcessing

Introduction This section provides general information to use theChargeSwitchgDNA Buccal Cell Kits (Catalog nos.CS11020-10 and CS11021-10) to process large numbers ofsamples in 96-well format using an automated liquidhandling robot. If you wish to process small numbers ofsamples individually, see General Information IndividualSamples, page 5.

HardwareRequirements

The ChargeSwitchchemistry is ideal for purification ofDNA using a liquid handling robot, avoiding the need forcentrifugation steps or the use of ethanol or chaotropic salts.You will need to have the following hardware to performautomated processing of buccal swabs using one of theChargeSwitchgDNA Buccal Cell Kits:

Any liquid handling robotic workstation with a gripperarm

Appropriate tips for liquid dispensing and aspiration(see below for factors to consider)

96-Well Magnetic Separator (see page 15)

Shaker

Incubator, heat block, or water bath for heating samples

96 x 2 ml deep well plate(s) (Greiner, Catalog no. 780270or Abgene, Catalog no. AB-0932)

96 x 300 l U-Bottomed microtiter plate (Greiner,Catalog no. 650201)

For an example of how to set up the deck, see page 16.

Tip Selection You may use any tips of choice to dispense and aspirateliquid during the purification procedure. Consider thefollowing factors when choosing an appropriate tip to use.

Fixed vs. disposable tips

Tip size vs. head size

Conductive or non-conductive

Sterile or non-sterile

Filtered or non-filtered


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General Information Automated SampleProcessing, continued

96-WellMagneticSeparator

The 96-Well Magnetic Separator available from Invitrogen(Catalog no. CS15096) is a magnetic separation rack that canhold up to 96 samples in a deep well plate. The deep wellplate fits onto the magnetic base station, associating thearray of 24 neodymium magnets with the samples for on themagnet and off the magnet sample processing (see figures

below). For more information, see www.invitrogen.comorcall Technical Service (page 30).


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Deck Set Up Once you have the required hardware, you will need toconfigure the deck of your liquid handling robotappropriately to process samples. You may use any suitableconfiguration of your choice. An example is provided below.

Location Trough Contents Plate

1 96-well Deep Well plate #1

2 Lysis Mix (i.e.ChargeSwitchLysisBuffer (L11) + Proteinase K)

3 Purification Mix (i.e.ChargeSwitchPurification Buffer (N6) +ChargeSwitchMagnetic Beads)

4 ChargeSwitchWash Buffer (W12)

5 ChargeSwitchElution Buffer (E5)

6

7 96-well Deep Well plate #2

8 Waste

9 96-well Magnetic Separator

10 Shaker

11 96-well Sample Tray

12 ChargeSwitchLysis Buffer (L11)

13 96-well U-bottomed microtiterplate (for final elution)


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Primary LiquidHandlingParameters

The table below lists the primary liquid handlingparameters required to isolate DNA using the automatedprotocol. Use the parameters and guidelines provided, aswell as the protocol on pages 21-22 to program your robot.

Parameter Aim Guidelines

[Magnetic BeadPreparation]

To resuspend beadsprior to mixing withsolution

Only required once

Beads stay in suspension for up to45 minutes

[Mixing #1] Used to mix beads orbead/DNA pelletwith buffer

Aspirate/dispense at 400-500 l

Aspirate/dispense position fixed 1-2 mmabove the well bottom

Use tips/volume setting at 80 l volume

[Dispense liquid] Normal liquidparameters for adding

a reagent to each well

Aspirate/dispense at 300-400 l

Use multi-dispense if appropriate to save

time

[Transfersupernatant towaste]

To remove anddiscard supernatant

Aspirate slowly at 50-100 l/second

Aspirate off the entire liquid volume usingliquid detect and tracking or setting fixedheight 1 mm above the well bottom

Do not disturb pellet

Dispense to waste

[Transfer

supernatant toanother plate]

To transfer

supernatant toanother plate

Aspirate slowly at 50-100 l/second

Aspirate off the entire liquid volume usingliquid detect and tracking or setting fixedheight 1 mm above the well bottom

Do not disturb pellet

Dispense slowly at 50-100 l/second

Avoid splashing

[Final DNAElution]

To dispense the eluatecontaining DNA

Dispense at 10 l/second

Aspirate from position fixed 1 mm abovethe well bottom

Avoid bead carry-over

Dispense into new plate at 2 mm above thewell bottom

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Automated Genomic DNA Isolation

Introduction This section provides a general protocol for automated

isolation of genomic DNA from human buccal cell swabs ina 96-well format using the ChargeSwitchgDNA Buccal CellKit (Catalog no. 11021-10). Use this general protocol todevelop the script for your liquid handling robot.

Note:If you are using the ChargeSwitchgDNA Normalized BuccalCell Kit (Catalog no. 11020-10), follow the protocol on pages 23-26.

The ChargeSwitchMagnetic Beads supplied in theChargeSwitchgDNA Buccal Cell Kit differ in concentrationfrom those supplied in the ChargeSwitchgDNANormalized Buccal Cell Kit. Use onlythe ChargeSwitchMagnetic Beads supplied in the ChargeSwitchgDNABuccal Cell Kit in this protocol.

MaterialsNeeded


Liquid handling robot configured to process samples in96-well plates

Buccal swabs

96 x 2 ml deep well plates

96 x 300 l U-bottomed microtiter plate

Optional: 96 x 2 ml glass-filled, polypropylene,UnifilterMicroplate (Whatman, Catalog no. 7720-7235;see the next page)



Proteinase K



ChargeSwitchWash Buffer (W12)

ChargeSwitchElution Buffer (E5) or TE Buffer (not

supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)


Unifilteris a registered trademark of Whatman

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Automated Genomic DNA Isolation,continued

ImportantGuidelines

To maximize DNA yield, follow these recommendationswhen processing your samples:

Ensure that the robotic tips enter the wells of the plateswithout interfering with the pellet of beads.

When removing supernatant, leave samples on the 96-Well Magnetic Separator and aspirate slowly to ensurethat the pellet of beads is not disturbed.

When resuspending pelleted ChargeSwitchMagneticBeads, make sure that all beads are fully resuspended tomaximize DNA recovery.

To maximize DNA yield, make sure that all WashBuffer is removed before elution.

To maximize DNA yield, make sure that the beads arefully resuspended during the elution step.

LysateVolume

The first step of the genomic DNA isolation protocolrequires addition of Lysis Mix to the sample. You may addeither 1 ml or 1.4 ml of Lysis Mix to the sample. Note thatsome of the Lysis Mix may be absorbed by the swab,resulting in a lower volume of lysate being available forpurification. To maximize DNA yield, we recommend using1.4 ml of Lysis Mix (Step 2, page 21). To prepare Lysis Mix,see the next page.

Using a FilterPlate

To maximize recovery volume and minimize contaminanttransfer during lysate preparation, you may prepare lysatesin a 96 x 2 ml filter plate (see Steps 1-2, page 21), thendirectly filter the supernatant into a 96 x 2 ml deep wellplate to perform the remainder of the purificationprocedure. We recommend using the 96 x 2 ml glass-filledpolypropylene UnifilterMicroplate from Whatman

(Catalog no. 7720-7235). Other 96 x 2 ml filter plates aresuitable.


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BeforeStarting


Prepare Lysis Mix: For each sample, mix 1 ml of

ChargeSwitchLysis Buffer (L11) and 10 l ofProteinase K to prepare the Lysis Mix. If lysing in 1.4 mlvolume, mix 1.4 ml of ChargeSwitchLysis Buffer (L11)and 10 l of Proteinase K for each sample. Scale up thevolume of reagents used (based on number of samples)to prepare a master mix.

Prepare Purification Mix: For each sample, mix 100 l

of ChargeSwitchPurification Buffer (N5) and 40 l ofChargeSwitchMagnetic Beads to prepare thePurification Mix (make sure that the beads arethoroughly resuspended). If lysing in 1.4 ml volume,mix 140 l of ChargeSwitchPurification Buffer (N5)and 40 l of ChargeSwitchMagnetic Beads for eachsample. Scale up the volume of reagents used (based on

number of samples) to prepare a master mix.


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AutomatedProtocol

Follow this protocol to isolate genomic DNA from buccalswabs. The volumes given are on a per sample basis.

1. Start with 96 buccal cell samples in a 96 x 2 ml deep wellplate or 96 x 2 ml filter plate.

2. Add 1 ml (or 1.4 ml) of Lysis Mix (see previous page) and

incubate at 37C for 20 minutes (use a heating block).

Note:Optimal incubation parameters (i.e.time) vary depending

on the sample and automation plasticware, and should bedetermined empirically.

3. After incubation, transfer or filter as appropriate, asmuch of the lysate as possible to a 96 x 2 ml deep wellplate, without interfering with the samples.

4. Add 140 l (or 180 l if 1.4 ml of Lysis Mix used) ofPurification Mix (see previous page; make sure that the

beads are thoroughly resuspended).

5. Shake at medium fast speed (e.g.pulse, 10 seconds) toevenly distribute the magnetic beads within the solution.

6. Wait for 10 seconds.

7. Move samples to the 96-Well Magnetic Separator.

8. Wait for 60-90 seconds.

9. Slowly aspirate all of the supernatant and discard,leaving behind the pellet of beads.

10. While samples are still on the 96-Well MagneticSeparator, add 1 ml of ChargeSwitchWash Buffer(W12).

11. Wait for 60 seconds or until the beads have formed atight pellet.


13. While samples are still on the 96-Well MagneticSeparator, add 1 ml of ChargeSwitchWash Buffer(W12).


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AutomatedProtocol,continued



16. Move samples to the shaker.

17. Add 150 l of ChargeSwitchElution Buffer (E5).

Note:You may vary elution volume depending on your needs.

Do not elute in volumes < 60 l as the DNA may not completely

elute from the beads.

18. Shake rapidly for 1-2 minutes to completely disperse thebeads within the solution.


20. Wait for 1 minute.

21. Slowly aspirate supernatant containing the DNAto a

96 x 300 l U-bottomed microtiter plate.

Storing DNA Store the purified DNA at -20C or use immediately fordownstream applications such as PCR. Avoid repeatedlyfreezing and thawing DNA.


To quantitate yield of your DNA, use the Quant-iTDNAAssay Kit, High Sensitivity (Catalog no. Q33120). For more

information, see page 13.

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Automated Genomic DNA Isolation Normalized Kit

Introduction This section provides a general protocol for automatedisolation of genomic DNA from human buccal cell swabs ina 96-well format using the ChargeSwitchgDNANormalized Buccal Cell Kit (Catalog no. 11020-10). Use thisgeneral protocol to develop the script for your liquidhandling robot.

Note:If you are using the ChargeSwitchgDNA Buccal Cell Kit(Catalog no. 11021-10), follow the guidelines and protocol on

pages 18-22.

The ChargeSwitchMagnetic Beads supplied in theChargeSwitchgDNA Normalized Buccal Cell Kit differ inconcentration from those supplied in the ChargeSwitchgDNA Buccal Cell Kit. Use onlythe ChargeSwitchMagnetic Beads supplied in the ChargeSwitchgDNANormalized Buccal Cell Kit in this protocol.

MaterialsNeeded


Liquid handling robot configured to process samples in96-well plates

Buccal swabs

96 x 2 ml deep well plates

96 x 300 l U-bottomed microtiter plate

Optional: 96 x 2 ml glass-filled, polypropylene,UnifilterMicroplate (Whatman, Catalog no. 7720-7235;see the next page)



Proteinase K



ChargeSwitchWash Buffer (W12)

ChargeSwitchElution Buffer (E5) or TE Buffer (notsupplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)


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Automated Genomic DNA Isolation Normalized Kit, continued

ImportantGuidelines

To maximize DNA yield, follow these recommendationswhen processing your samples:

Ensure that the robotic tips enter the wells of the plateswithout interfering with the pellet of beads.

When removing supernatant, leave samples on the 96-Well Magnetic Separator and aspirate slowly to ensurethat the pellet of beads is not disturbed.

When resuspending pelleted ChargeSwitchMagneticBeads, make sure that all beads are fully resuspended tomaximize DNA recovery.

To maximize DNA yield, make sure that all WashBuffer is removed before elution.

To maximize DNA yield, make sure that the beads arefully resuspended during the elution step.

Using a FilterPlate

Some of the Lysis Mix may be absorbed by the sample,resulting in a lower volume of lysate being available forpurification. To maximize recovery volume and minimizecontaminant transfer during lysate preparation, you mayprepare lysates in a 96 x 2 ml filter plate (see Steps 1-2,page 25), then directly filter the supernatant into a 96 x 2 mldeep well plate to perform the remainder of the purificationprocedure. We recommend using the 96 x 2 ml glass-filled

polypropylene UnifilterMicroplate from Whatman(Catalog no. 7720-7235). Other 96 x 2 ml filter plates aresuitable.


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BeforeStarting


Prepare Lysis Mix: For each sample, mix 1 ml of

ChargeSwitchLysis Buffer (L11) and 10 l ofProteinase K to prepare the Lysis Mix. Scale up thevolume of reagents used (based on number of samples)to prepare a master mix.

Prepare Purification Mix: For each sample, mix 100 l

of ChargeSwitch

Purification Buffer (N5) and 40 l ofChargeSwitchMagnetic Beads to prepare thePurification Mix (make sure that the beads arethoroughly resuspended). Scale up the volume ofreagents used (based on number of samples) to preparea master mix.

Automated

Protocol

Follow this protocol to isolate genomic DNA from buccal

swabs. The volumes given are on a per sample basis.1. Start with 96 buccal cell samples in a 96 x 2 ml deep well

plate or 96 x 2 ml filter plate.

2. Add 1 ml of Lysis Mix (see above) and incubate at 37Cfor 20 minutes (use a heating block).

Note:Optimal incubation parameters (i.e.time) vary dependingon the sample and automation plasticware, and should bedetermined empirically.

3. After incubation, transfer or filter as appropriate, asmuch of the lysate as possible to a 96 x 2 ml deep wellplate, without interfering with the samples.

4. Add 140 l of Purification Mix (see above; make sure thatthe beads are thoroughly resuspended).


6. Wait for 10 seconds.





25


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AutomatedProtocol,continued

10. Remove samples from the 96-Well Magnetic Separator.

11. Add 500 l of ChargeSwitchWash Buffer (W12).





16. While samples are still on the 96-Well Magnetic

Separator, add 500 l of ChargeSwitchWash Buffer(W12).



19. Move samples to the shaker.

20. Add 150 l of ChargeSwitchElution Buffer (E5).

21. Shake rapidly for 1-2 minutes to completely disperse thebeads within the solution.


23. Wait for 1 minute.

24. Slowly aspirate supernatant containing the DNAto a

96 x 300 l U-bottomed microtiter plate.

Storing DNA Store the purified DNA at -20C or use immediately fordownstream applications such as PCR. Avoid repeatedlyfreezing and thawing DNA.


To quantitate the yield of your DNA, use the Quant-iT

DNA Assay Kit, High Sensitivity (Catalog no. Q33120). Formore information, see page 13.

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Troubleshooting

Introduction Refer to the table below to troubleshoot problems that you

may encounter when purifying genomic DNA with the kit.

Problem Cause Solution

Low DNA yield Incomplete lysis Be sure to add Proteinase Kduring lysis.

Increase the length of

incubation at 37C.

Poor quality ofstarting material Process samples immediatelyafter collection or store the

sample at 4C. Do not storesamples at room temperature as

bacteria and nucleases presentin the sample will degrade theDNA.

Insufficient amount ofChargeSwitch

Magnetic Beads added

Vortex the tube containing theChargeSwitchMagnetic

Beads to fully resuspend thebeads in solution beforepreparing the PurificationMix.

Before adding PurificationMix to your sample, makesure that the beads are fullyresuspended.

Pellet of beadsdisturbed or lostduring binding orwashing steps

Keep the sample in theMagnaRackor 96-WellMagnetic Separator whenremoving supernatant duringthe binding or washing steps.

Remove the supernatantwithout disturbing the pelletof beads by angling thepipette tip away from the

pellet.

Bubbles formedduring mixing steps

Make sure that the pipette tip issubmerged in the solutionduring mixing.


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Troubleshooting, continued


Low DNA yield,continued

Incomplete disso-ciation of DNA fromthe ChargeSwitchMagnetic Beads

Perform additional mixing ofthe suspension of beads (bypipetting up and down).

Incorrect elutionconditions

After adding ChargeSwitchElution Buffer (E5) to thesample, pipet up and down tofully resuspend the magnetic

beads before incubation.

Do not use water for elution.Use ChargeSwitchElutionBuffer (E5) or TE, pH 8.5.

Lysate mixed toovigorously or smallpipette tips usedduring mixing

Use the appropriate pipettetip set to a volume lower thanthe total volume of solution inthe sample.

Pipet up and down gently tomix.

No DNA recovered Water used for elution Do not use water for elution.The elution buffer musthave apH = 8.5-9.0 or the DNA willremain bound to theChargeSwitchMagnetic Beads.Use Elution Buffer (E5) or TE,

pH 8.5.ChargeSwitchMagnetic Beads storedor handledimproperly

Store beads at roomtemperature. Do not freeze the

beads as they will becomeirreparably damaged.

Make sure that the beads arein solution at all times and donot become dried. Dried

beads are non-functional.

Purification Mix didnot containChargeSwitchMagnetic Beads

Purification Mix should containChargeSwitchPurificationBuffer (N6) + ChargeSwitchMagnetic Beads.


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Troubleshooting, continued


DNA is degraded Buccal swabs stored atroom temperature

Process buccal swabsimmediately after collection or

store at 4C.

DNA yields varywidely betweensamples

Variability in samplecollection

DNA yields can vary dependingon swabbing technique, whetherthe donor is a high or lowshedder, and the type of swabused.

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Appendix

Technical Service

World WideWeb

Visit the Invitrogen Web Resource using your World WideWeb browser. At the site, you can:

Get the scoop on our hot new products and specialproduct offers

View and download vector maps and sequences

Download manuals in AdobeAcrobat(PDF) format

Explore our catalog with full color graphics

Obtain citations for Invitrogen products

Request catalog and product literature

Once connected to the Internet, launch your Web browser(Internet Explorer 5.0 or newer or Netscape 4.0 or newer),then enter the following location (or URL):

http://www.invitrogen.com

...and the program will connect directly. Click on underlinedtext or outlined graphics to explore. Don't forget to put a

bookmark at our site for easy reference!

Contact Us For more information or technical assistance, call, write, fax,or email. Additional international offices are listed on ourWeb page (www.invitrogen.com).

Corporate Headquarters:

European Headquarters:Invitrogen Corporation Invitrogen Ltd

1600 Faraday Avenue Inchinnan Business Park

Carlsbad, CA 92008 USA 3 Fountain Drive

Tel: 1 760 603 7200 Paisley PA4 9RF, UK

Tel (Toll Free): 1 800 955 6288 Tel: +44 (0) 141 814 6100

Fax: 1 760 602 6500 Tech Fax: +44 (0) 141 814 6117

E-mail:[email protected]

E-mail:[email protected]


30
http://www.invitrogen.com/http://www.invitrogen.com/mailto:[email protected]:[email protected]:[email protected]:[email protected]://www.invitrogen.com/http://www.invitrogen.com/


37/40

Technical Service, continued

MSDS

Requests

To request an MSDS, visit our Web site at

www.invitrogen.com. On the home page, go to TechnicalResources, select MSDS, and follow instructions on thepage.

LimitedWarranty

Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that everycustomer is 100% satisfied with our products and our service. If youshould have any questions or concerns about an Invitrogen productor service, contact our Technical Service Representatives.

Invitrogen warrants that all of its products will perform accordingto specifications stated on the certificate of analysis. The companywill replace, free of charge, any product that does not meet thosespecifications. This warranty limits Invitrogen Corporationsliability only to the cost of the product. No warranty is granted forproducts beyond their listed expiration date. No warranty isapplicable unless all product components are stored in accordancewith instructions. Invitrogen reserves the right to select themethod(s) used to analyze a product unless Invitrogen agrees to a

specified method in writing prior to acceptance of the order.Invitrogen makes every effort to ensure the accuracy of itspublications, but realizes that the occasional typographical or othererror is inevitable. Therefore Invitrogen makes no warranty of anykind regarding the contents of any publications or documentation. Ifyou discover an error in any of our publications, please report it toour Technical Service Representatives.

Invitrogen assumes no responsibility or liability for any special,incidental, indirect or consequential loss or damage whatsoever.The above limited warranty is sole and exclusive. No other

warranty is made, whether expressed or implied, including anywarranty of merchantability or fitness for a particular purpose.

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Purchaser Notification and ProductQualification

PurchaserNotification

Limited Use Label License No. 265: ChargeSwitchTechnology

The use of this product may be covered by European PatentNo. EP1036082B1 and foreign equivalents.

ProductQualification

Each kit is functionally tested to ensure conformance with themost current approved product specifications. Current

specifications consist of tests for:

Bead size, charge, and binding capacity

Nucleic acid quality and quantity

Buffer turbidity, volume, and absence of RNases andDNases

Kit packaging and labeling accuracy

For individual lot test results and more information, visit

www.invitrogen.com to download the Certificate of Analysis.

2005 Invitrogen Corporation. All rights reserved.

For research use only. Not intended for any animal or humantherapeutic or diagnostic use.

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Corporate Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, California 92008Tel: 1 760 603 7200Tel (Toll Free): 1 800 955 6288Fax: 1 760 603 7229Email: [email protected]

European Headquarters:Invitrogen Ltd

3 Fountain DriveInchinnan Business ParkPaisley PA4 9RF, UKTel (Free Phone Orders): 0800 269 210Tel (General Enquiries): 0800 5345 5345Fax: +44 (0) 141 814 6287Email: [email protected]

International Offices:Argentina 5411 4556 0844Australia 1 800 331 627Austria 0800 20 1087Belgium 0800 14894Brazil 0800 11 0575

Canada 800 263 6236China 10 6849 2578Denmark 80 30 17 40

France 0800 23 20 79Germany 0800 083 0902Hong Kong 2407 8450India 11 577 3282Italy 02 98 22 201

Japan 03 3663 7974The Netherlands 0800 099 3310New Zealand 0800 600 200Norway 00800 5456 5456

Spain & Portugal 900 181 461Sweden 020 26 34 52Switzerland 0800 848 800Taiwan 2 2651 6156UK 0800 838 380For other countries see our Web site

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Chargeswitch GDNA - Buccal - Manual

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