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The Invitrogen iPrep ChargeSwitch gDNA Buccal Cell Kits allow rapid and efficient purification of genomic DNA from human buccal swabs. After preparing the lysates, you may purify DNA in less than 15 minutes using the ChargeSwitch Technology. Depending on the kit used, samples may be handled individually or in an automated system using a liquid handling robot.
Intended Use for the Kits
The ChargeSwitch gDNA Buccal Cell Kits are designed to allow isolation of the following amounts of genomic DNA from human buccal cell swabs or pelleted cells from a mouthwash. Samples can be stored for up to 2 weeks at 4°C before processing without a noticeable loss in DNA yield or quality. The purified genomic DNA is suitable for use in downstream applications such as PCR.
Important: The DNA yield varies and is dependent on several factors including the technique of the person taking the swab, whether the donor is a high or low shedder, and the type of swab used.
Advantages
Use of the ChargeSwitch gDNA Buccal Cell Kits to isolate genomic DNA provides the following advantages:
System Specifications
Starting Material: | Human buccal swabs |
Elution Volume: | 150 µl |
DNA Yield: | 1-3 ng/µl in 150 µl (Normalized Buccal Cell Kit) or up to 6µg (Buccal Cell Kit) |
DNA Size: | Varies (depends on quality of starting material) |
The ChargeSwitch Technology
The ChargeSwitch Technology (CST) is a novel magnetic bead-based technology that provides a switchable surface charge dependent on the pH of the surrounding buffer to facilitate nucleic acid purification. In low pH conditions, the CST beads have a positive charge that binds the negatively charged nucleic acid backbone (see figure below). Proteins and other contaminants are not bound and are simply washed away in an aqueous wash buffer. To elute nucleic acids, the charge on the surface of the bead is neutralized by raising the pH to 8.5 using a low salt elution buffer (see figure below). Purified DNA elutes instantly into this elution buffer, and is ready for use in downstream applications.
ChargeSwitch Magnetic Bead Specifications
Bead Binding Capacity: | 5-10 µg genomic DNA per mg |
Bead Size: | < 1 µm |
Bead Concentration: | 25 mg/ml (Buccal Cell Kits only) 3.125 mg/ml (Normalized Buccal Cell Kits only) |
Storage Buffer: | 10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20 |
Automated Liquid Handling
Use of the ChargeSwitch gDNA Buccal Cell Kits has been demonstrated on the Thermo Scientific Tecan Genesis robotic workstation to purify DNA in a fully automated system from large numbers of buccal cell swabs in a 96-well format. Other liquid handling robots are suitable provided that each is equipped with a gripper arm, a 96-well magnetic separator, and other additional hardware. This manual provides general guidelines and a protocol that may be used to develop a script for your robot. Genesis® is a registered trademark of Tecan AG Group.
Introduction
This section provides general information needed to use the ChargeSwitch gDNA Buccal Cell Kits (Cat. Nos. CS11020 or CS11021) to process individual samples. If you are using a liquid handling robot to process large numbers of samples, see General Information – Automated Sample Processing.
User Supplied Materials
In addition to the reagents supplied with the kit, you need to have the following materials on hand before beginning:
MagnaRack
The Invitrogen MagnaRack (Cat. No. CS15000) is a two-piece magnetic separation rack for use in protocols with magnetic beads, and consists of a magnetic base station and a removable tube rack. The tube rack can hold up to 24 microcentrifuge tubes. The tube rack fits onto the magnetic base station in two different positions, associating the row of 12 neodymium magnets with a single row of 12 tubes for simple ‘on the magnet’ and ‘off the magnet’ sample processing (see figure below).
Safety Information
Follow the safety guidelines below when using the ChargeSwitch gDNA Buccal Cell Kits.
Handling the ChargeSwitch Magnetic Beads
Follow the guidelines below when handling the ChargeSwitch magnetic beads.
Elution Buffer
Invitrogen ChargeSwitch Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) is supplied with the kit for eluting the DNA from the Invitrogen ChargeSwitch Magnetic Beads. For best results, use Elution Buffer (E5) to elute the DNA. Alternatively, TE Buffer, pH 8.5-9.0 is acceptable. Note that the pH must be between 8.5-9.0 otherwise the DNA will not elute. Do not use water for elution.
The protocol suggests eluting the genomic DNA in 150 µl of ChargeSwitch Elution Buffer (E5). When using the ChargeSwitch gDNA Buccal Cell Kit, you may vary the amount of ChargeSwitch Elution Buffer (E5) used to obtain genomic DNA in the desired final concentration. For best results, always use a volume of ChargeSwitch Elution Buffer (E5) that is equal to or greater than the volume of ChargeSwitch Magnetic Beads used in the protocol. If the volume of ChargeSwitch Elution Buffer (E5) is lower than the volume of beads used, DNA elution is incomplete. You may need to perform a second elution to recover all DNA.
Important: When using the ChargeSwitch gDNA Normalized Buccal Cell Kit, elute the genomic DNA in 150 µl of ChargeSwitch Elution Buffer (E5). Do not vary the amount of ChargeSwitch Elution Buffer (E5) used otherwise the yield will no longer be normalized at 1-3 ng/µl
Introduction
This section provides guidelines and instructions to isolate genomic DNA from human buccal swabs or pelleted cells from a mouthwash using the reagents supplied in the kit. Note that the protocol is optimized for efficient purification of DNA from small sample volumes. Depending on the volume of your sample, some further optimization of the protocol may be required.
Starting Material
Use this procedure to isolate genomic DNA from human buccal cell swabs or pelleted cells from a mouthwash. Process samples immediately after collection or store at 4°C for up to 2 weeks. Do not store unprocessed samples at room temperature as buccal swabs contain bacteria and nucleases that will degrade DNA.
The ChargeSwitch® Magnetic Beads supplied in the ChargeSwitch gDNA Buccal Cell Kit differ in concentration from those supplied in the ChargeSwitch gDNA Normalized Buccal Cell Kit. Although the purification protocol is identical for both kits, you must use the ChargeSwitch Magnetic Beads supplied with each kit to obtain the DNA yields specified.
Do not substitute ChargeSwitch Magnetic Beads provided in the ChargeSwitch gDNA Buccal Cell Kit for those in the ChargeSwitch gDNA Normalized Buccal Cell Kit.
Introduction
This section provides general information to use the ChargeSwitch gDNA Buccal Cell Kits (Cat. Nos. CS11020-10 and CS11021-10) to process large numbers of samples in 96-well format using an automated liquid handling robot. If you wish to process small numbers of samples individually, see General Information – Individual Samples., page 5.
Hardware Requirements
The ChargeSwitch chemistry is ideal for purification of DNA using a liquid handling robot, avoiding the need for centrifugation steps or the use of ethanol or chaotropic salts. You will need to have the following hardware to perform automated processing of buccal swabs using one of the ChargeSwitch gDNA Buccal Cell Kits:
Tip Selection
You may use any tips of choice to dispense and aspirate liquid during the purification procedure. Consider the following factors when choosing an appropriate tip to use.
96-Well Magnetic Separator
The 96-Well Magnetic Separator (Cat. No. CS15096) is a magnetic separation rack that can hold up to 96 samples in a deep well plate. The deep well plate fits onto the magnetic base station, associating the array of 24 neodymium magnets with the samples for ‘on the magnet’ and ‘off the magnet’ sample processing (see figures below).
Deck Set Up
Once you have the required hardware, you will need to configure the deck of your liquid handling robot appropriately to process samples. You may use any suitable configuration of your choice. An example is provided below.
Location | Trough Contents | Plate |
1
|
96-well Deep Well plate #1
| |
2
|
Lysis Mix (
i.e. ChargeSwitch Lysis Buffer (L11) + Proteinase K)
| |
3
|
Purification Mix (
i.e. ChargeSwitch Purification Buffer (N6) + ChargeSwitch Magnetic Beads)
| |
4
|
ChargeSwitch Wash Buffer (W12)
| |
5
|
ChargeSwitch Elution 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
|
ChargeSwitch Lysis Buffer (L11)
| |
13
|
96-well U-bottomed microtiter plate (for final elution)
|
Primary Liquid Handling Parameters
The table below lists the primary liquid handling parameters required to isolate DNA using the automated protocol. Use the parameters and guidelines provided to program your robot.
.
Parameter | Aim | Guidelines |
[Magnetic Bead Preparation]
|
To resuspend beads prior to mixing with solution
|
|
[Mixing #1]
|
Used to mix beads or bead/DNA pellet with buffer
|
|
[Dispense liquid]
|
Normal liquid parameters for adding a reagent to each well
|
|
[Transfer supernatant to waste]
|
To remove and discard supernatant
|
|
[Transfer supernatant to another plate]
|
To transfer supernatant to another plate
|
|
[Final DNA Elution]
|
To dispense the eluate containing DNA
|
|
Introduction
This section provides a general protocol for automated isolation of genomic DNA from human buccal cell swabs in a 96-well format using the ChargeSwitch gDNA Buccal Cell Kit (Cat. No. 11021-10). Use this general protocol to develop the script for your liquid handling robot.
Note: If you are using the ChargeSwitch gDNA Normalized Buccal Cell Kit (Cat. No. 11020-10), see Automated Extraction Normalized DNA Buccal Kit
The ChargeSwitch Magnetic Beads supplied in the ChargeSwitch gDNA Buccal Cell Kit differ in concentration from those supplied in the ChargeSwitch gDNA Normalized Buccal Cell Kit. Use only the ChargeSwitch Magnetic Beads supplied in the ChargeSwitch gDNA Buccal Cell Kit in this protocol.
Materials Needed
Have the following materials on hand before beginning:
Components Supplied with the Kit includes Invitrogen products such as
Unifilter is a registered trademark of Whatman
Important Guidelines
To maximize DNA yield, follow these recommendations when processing your samples:
Lysate Volume
The first step of the genomic DNA isolation protocol requires addition of Lysis Mix to the sample. You may add either 1 ml or 1.4 ml of Lysis Mix to the sample. Note that some of the Lysis Mix may be absorbed by the swab, resulting in a lower volume of lysate being available for purification. To maximize DNA yield, we recommend using 1.4 ml of Lysis Mix (Step 2).
Using a Filter Plate
To maximize recovery volume and minimize contaminant transfer during lysate preparation, you may prepare lysates in a 96 x 2 ml filter plate, then directly filter the supernatant into a 96 x 2 ml deep well plate to perform the remainder of the purification procedure. We recommend using the 96 x 2 ml glass-filled polypropylene Unifilter®Microplate from Whatman (Catalog no. 7720-7235). Other 96 x 2 ml filter plates are suitable.
Before Starting
Perform the following before beginning:
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 (or 1.4 ml) of Lysis Mix and incubate at 37°C for 20 minutes (use a heating block).
Note: Optimal incubation parameters (i.e. time) vary depending on the sample and automation plasticware, and should be determined empirically.
3. After incubation, transfer or filter as appropriate, as much of the lysate as possible to a 96 x 2 ml deep well plate, without interfering with the samples.
4. Add 140 µl (or 180 µl if 1.4 ml of Lysis Mix used) of Purification Mix ( (make sure that the beads are thoroughly resuspended).
5. Shake at medium fast speed (e.g. pulse, 10 seconds) to evenly 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 Magnetic Separator, add 1 ml of ChargeSwitch Wash Buffer (W12).
11. Wait for 60 seconds or until the beads have formed a tight pellet.
12. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.
13. While samples are still on the 96-Well Magnetic Separator, add 1 ml of ChargeSwitch Wash Buffer (W12).
14. Wait for 30-60 seconds.
15. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.
16. Move samples to the shaker.
17. Add 150 µl of ChargeSwitch Elution Buffer (E5).
Note: You may vary elution volume depending on your needs. Do not elute in volumes < 60 µl as the DNA may not completely elute from the beads.
18. Shake rapidly for 1-2 minutes to completely disperse the beads within the solution.
19. Move samples to the 96-Well Magnetic Separator.
20. Wait for 1 minute.
21. Slowly aspirate supernatant containing the DNA to a 96 x 300 µl U-bottomed microtiter plate.
Storing DNA
Store the purified DNA at -20C or use immediately for downstream applications such as PCR. Avoid repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate yield of your DNA, use the Invitrogen Quant-iT DNA Assay Kit, High Sensitivity (Cat. No. Q33120).
Have the following materials on hand before beginning:
Components Supplied with the Kit includes Invitrogen products such as
Before Starting
Perform the following before beginning:
Preparing the Lysate
Follow the procedure below to prepare a lysate from the human buccal cell swab.
Binding DNA
Follow the procedure below to bind the DNA to the ChargeSwitch Magnetic Beads.
Washing DNA
Eluting DNA
Storing DNA
Store the purified DNA at -20C or use immediately for PCR or other appropriate downstream application. Avoid repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate yield of your DNA, we recommend using the Quant-iT DNA Assay Kit, High Sensitivity (Cat. No. Q33120). This kit contains a state-of-the-art quantitation reagent, pre-diluted standards, and a pre-made buffer to allow sensitive and accurate fluorescence-based quantitation of dsDNA.
Introduction
This section provides a general protocol for automated isolation of genomic DNA from human buccal cell swabs in a 96-well format using the ChargeSwitch gDNA Normalized Buccal Cell Kit (Cat. No. 11020-10). Use this general protocol to develop the script for your liquid handling robot. Note: If you are using the ChargeSwitch gDNA Buccal Cell Kit (Cat. No. 11021-10), follow the guidelines and protocol below.
The ChargeSwitch Magnetic Beads supplied in the ChargeSwitch gDNA Normalized Buccal Cell Kit differ in concentration from those supplied in the ChargeSwitch gDNA Buccal Cell Kit. Use only the ChargeSwitch Magnetic Beads supplied in the ChargeSwitch gDNA Normalized Buccal Cell Kit in this protocol.
Materials Needed
Have the following materials on hand before beginning:
Optional: 96 x 2 ml glass-filled, polypropylene, Unifilter® Microplate (Whatman, Cat. No. 7720-7235)
Components Supplied with the Kit includes Invitrogen products such as
Important Guidelines
To maximize DNA yield, follow these recommendations when processing your samples:
Using a Filter Plate
Some of the Lysis Mix may be absorbed by the sample, resulting in a lower volume of lysate being available for purification. To maximize recovery volume and minimize contaminant transfer during lysate preparation, you may prepare lysates in a 96 x 2 ml filter plate, then directly filter the supernatant into a 96 x 2 ml deep well plate to perform the remainder of the purification procedure. We recommend using the 96 x 2 ml glass-filled polypropylene Unifilter® Microplate from Whatman (Cat. No. 7720-7235). Other 96 x 2 ml filter plates are suitable.
Before Starting
Perform the following before beginning:
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 37°C for 20 minutes (use a heating block).
Note: Optimal incubation parameters (i.e. time) vary depending on the sample and automation plasticware, and should be determined empirically.
3. After incubation, transfer or filter as appropriate, as much of the lysate as possible to a 96 x 2 ml deep well plate, without interfering with the samples.
4. Add 140 µl of Purification Mix (see above; make sure that the beads are thoroughly resuspended).
5. Shake at medium fast speed (e.g. pulse, 10 seconds) to evenly 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. Remove samples from the 96-Well Magnetic Separator.
11. Add 500 µl of ChargeSwitch Wash Buffer (W12).
12. Shake at medium fast speed (e.g. pulse, 10 seconds) to evenly distribute the magnetic beads within the solution.
13. Move samples to the 96-Well Magnetic Separator.
14. Wait for 60-90 seconds.
15. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.
16. While samples are still on the 96-Well Magnetic Separator, add 500 µl of ChargeSwitch Wash Buffer (W12).
17. Wait for 30-60 seconds.
18. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.
19. Move samples to the shaker.
20. Add 150 µl of ChargeSwitc Elution Buffer (E5).
21. Shake rapidly for 1-2 minutes to completely disperse the beads within the solution.
22. Move samples to the 96-Well Magnetic Separator.
23. Wait for 1 minute.
24. Slowly aspirate supernatant containing the DNA to a 96 x 300 µl U-bottomed microtiter plate.
Storing DNA
Store the purified DNA at -20°C or use immediately for downstream applications such as PCR. Avoid repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate the yield of your DNA, use the Quant-iT DNA Assay Kit, High Sensitivity (Cat. No. Q33120).
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
|
|
Poor quality of starting material
|
Process samples immediately after collection or store the sample at 4°C. Do not store samples at room temperature as bacteria and nucleases present in the sample will degrade the DNA.
| |
Insufficient amount of ChargeSwitc Magnetic Beads added
|
| |
Pellet of beads disturbed or lost during binding or washing steps
|
| |
Bubbles formed during mixing steps
|
Make sure that the pipette tip is submerged in the solution during mixing.
| |
Problem | Cause | Solution |
Low DNA yield, continued
|
Incomplete disso-ciation of DNA from the ChargeSwitch Magnetic Beads
|
Perform additional mixing of the suspension of beads (by pipetting up and down).
|
Incorrect elution conditions
|
| |
Lysate mixed too vigorously or small pipette tips used during mixing
|
| |
No DNA recovered
|
Water used for elution
|
Do not use water for elution. The elution buffer
must have a pH = 8.5-9.0 or the DNA will remain bound to the ChargeSwitch Magnetic Beads. Use Elution Buffer (E5) or TE, pH 8.5.
|
ChargeSwitch Magnetic Beads stored or handled improperly
|
| |
Purification Mix did not contain ChargeSwitch Magnetic Beads
|
Purification Mix should contain ChargeSwitch Purification Buffer (N6) + ChargeSwitch Magnetic Beads.
| |
Problem | Cause | Solution |
DNA is degraded
|
Buccal swabs stored at room temperature
|
Process buccal swabs immediately after collection or store at 4°C.
|
DNA yields vary widely between samples
|
Variability in sample collection
|
DNA yields can vary depending on swabbing technique, whether the donor is a high or low shedder, and the type of swab used.
|
For Research Use Only. Not for use in diagnostic procedures.