实验概要
The iPrep™ GeneCatcher™ gDNA Blood Kit allows rapid and automated extraction of genomic DNA (gDNA) from human blood including archived or poorly stored blood samples. Genomic DNA is extracted from blood samples using the GeneCatcher™ Technology and iPrep™ Purification Instrument within 45 minutes without the use of centrifugation. For more information on the GeneCatcher™ Technology, see below. The purified genomic DNA is suitable for use in downstream applications including PCR and restriction enzyme digestion.
The iPrep™ GeneCatcher™ gDNA Blood Kit is designed for use with the iPrep™ Purification Instrument. The iPrep™ Purification Instrument is a benchtop, automated nucleic acid purification instrument with integrated Magnetic and Syringe Unit capable of purifying nucleic acids from up to 13 samples (12 samples 1 positive control) using a magnetic bead-based technology.
实验原理
The iPrep™ GeneCatcher™ gDNA Blood Kit combines the powerful GeneCatcher™ technology (next page) with the speed and convenience of the iPrep™ Instrument to allow automated purification of high-quality DNA from up to 13 samples (12 samples 1 positive control) within 45 minutes. Purification is achieved using a simple magnetic bead based purification procedure, and avoids the use centrifuges or vacuum manifolds. The lysate is mixed with GeneCatcher™ Magnetic Beads for subsequent DNA binding to the beads. The DNA-bound magnetic beads are separated from the lysate using magnetic separation. Any residual proteins are digested with the Protease. The beads are thoroughly washed with Wash Buffers to remove contaminants. The genomic DNA is then eluted in Elution Buffer (E5).
The GeneCatcher™ Technology is a novel magnetic bead-based technology that is designed to work on a wide range of blood samples including archived or poorly stored blood samples to facilitate genomic DNA purification.
Step 1–DNA Capture–Cells are lysed and crude DNA is captured on magnetic beads leaving most of the cell debris and protein behind in solution.
Step 2–DNA Purification–Any residual protein is digested using the Protease and then washed away to leave pure intact DNA.
Step 3–Elution–The pure DNA is then eluted into a small volume ready for use in any downstream applications.
The GeneCatcher™ gDNA Blood Kits provide a flexible solution for large-scale genotyping and biobanking projects due to the following advantages:
Uses a magnetic bead-based technology to isolate genomic DNA without the need for centrifugation or vacuum manifolds
Rapid and automated purification of genomic DNA within 45 minutes from a wide range of blood samples including difficult blood samples using the iPrep™ Instrument
Pre-filled reagent cartridges provide easy set up and consistent results
Minimal contamination with RNA
Purified genomic DNA demonstrates improved downstream performance in applications including PCR, restriction enzyme digestion, and Southern blotting.
实验步骤
Experienced Users Procedure
Introduction
This quick reference sheet is included for experienced users of the iPrep™ GeneCatcher™ gDNA Blood Kit. For more details, refer to details below the quick reference.
Procedure
Purification Protocol
Mix the fresh blood samples or thaw frozen blood samples.
Open the iPrep™ Card Slot and insert the iPrep™ gDNA Blood and Tissue Card, version 2.0 in the slot (arrow on the card is at the top and card label is facing your left side).
Turn ON the iPrep™ Instrument using the power switch on the left side of the instrument. The digital display shows the version for the iPrep™ which changes in few seconds to display the Main menu.
Press Start to run a protocol.
Press 2 to select the Blood protocol.
Open the iPrep™ door and remove iPrep™ Racks to set up the platform.
Remove the iPrep™ gDNA Blood Cartridges from the box. To collect any solution from the foil, tap the cartridge to deposit the solution at the bottom of the tube.
Insert one iPrep™ Sample and Elution Tube in position 11 for each of the iPrep™ gDNA Blood Cartridge that is used.
Load the cartridges on the iPrep™ Cartridge Rack and insert the loaded rack on to the iPrep™ Platform.
Load the iPrep™ Tip and Tube Rack as follows:
l Load the first row (labeled as E) with 1-13 elution tubes without caps
l Keep the second row (labeled as T1) empty
l Load the third row (labeled as T2) with iPrep™ Tips in the iPrep™ Tip Holders
l Load the fourth row (labeled as S) with sample tubes without caps and add 350 μl blood samples to the sample tubes
Read the sample and elution tube barcodes, if needed.
Insert the iPrep™ Tip and Tube Rack on to the iPrep™ Platform.
Close the iPrep™ Door. Press 1 to continue.
Select the appropriate elution volume on the display.
Press Start. The automated purification protocol begins and various steps of the protocol including the approximate time remaining are displayed on the digital display.
At the end of the run, the instrument beeps briefly and the digital display shows Protocol Finished for 10 seconds. The Main menu appears after 10 seconds.
Open the instrument door.
Remove and cap the elution tubes containing the purified nucleic acid. Store the purified gDNA at 4ºC (short-term) or aliquot and store at -20ºC (long-term).
Discard the used cartridges, tips, and tubes into biohazard waste. Do not reuse the cartridges.
To purify more samples using the same iPrep™ Blood and Tissue Card, load the racks with new cartridges, tips, tubes, and samples, and start the protocol as described.
If you are not using the instrument, close the instrument door and turn the power switch to OFF.
Remove the iPrep™ Blood Card and store card in the box.
iPrep™ Purification Instrument
Introduction
The iPrep™ Purification Instrument is a benchtop, automated nucleic acid purification instrument with integrated Magnetic and Syringe Unit capable of purifying nucleic acids from up to 12 samples and one positive control. Each iPrep™ Instrument consists of the Magnetic and Syringe Unit, and a platform. A pre-programmed iPrep™ Protocol Card controls the purification parameters such as buffer volumes, mixing steps, and incubation time. For more details on the iPrep™ Purification Instrument, see the manual supplied with the instrument.
iPrep™ Reaction Cartridge
The iPrep™ Reaction Cartridges are supplied with the iPrep™ Kits and are designed to fit onto the iPrep™ Cartridge Rack in only one orientation. Each cartridge is pre-filled with reagents required for the iPrep™ GeneCatcher™ gDNA Blood protocol.
Each cartridge has 12 positions with 10 sealed wells and two heating positions (position 12 with an empty well and position 11 to add an empty or reagent filled tube). For the iPrep™ GeneCatcher™ gDNA Blood Kit, positions 1-10 contain wells filled with reagents.
Cartridge Specifications:
Material: Polypropylene cartridge sealed with laminated aluminum foil
Max Volume: 1000 μl/well
Dimension: 5.9 inches (l) x 1.2 inches (w) x 0.7 inches (d)
iPrep™ Tips and Tip Holders
The iPrep™ Tips and Tip Holders are included with the iPrep™ Kits and are placed on the iPrep™ Tip and Tube Rack as described beolw. While assembling the tips on the rack, insert the iPrep™ Tips into the iPrep™ Tip Holders using gloved hands. Always use the tips with the holders to prevent any contamination.
Tip Specifications:
Tip Material: Polypropylene with filter barriers
Tip Holder Material: Polypropylene
Volume: 5-1000 μl
Tip Dimension: 3.9 inches (l) x 0.43 inches (d)
iPrep™ Tubes
Three sets of iPrep™ Tubes are required for the purification protocol. The iPrep™ Sample and Elution Tubes are included with each iPrep™ Kit and placed on the iPrep™ Tip and Tube Rack as described on below.
Tube Specifications:
Material: Polypropylene
Capacity: 1. 5 ml
Style: Tubes with caps
Dimensions: 1.7 inches (l) x 0.4 inches (d)
iPrep™ gDNA Blood and Tissue Card
To use the iPrep™ GeneCatcher™ gDNA Blood Kit with the iPrep™ Purification Instrument, you need to purchase the iPrep™ gDNA Blood and Tissue Card, version 2.0. The iPrep™ gDNA Blood and Tissue Card is pre-programmed with the purification protocol (for blood and tissue) that directs the volume of reagents used and incubation time.
Always store the card in the box, protected from light.
To avoid damaging the card:
Do not drop or bend the card
Do not wipe or clean the card using volatile chemicals such as alcohol or equivalent
Do not expose the card to water
iPrep™ Platform
The platform on the iPrep™ Instrument allows the placement of iPrep™ Tip and Tube Rack, and iPrep™ Cartridge Rack that are filled with plastic disposables and reagent cartridges required for the purification protocol.
Methods
General Information
User Supplied Materials
In addition to the reagents supplied with the kit, you also need the following materials and instrumentation:
iPrep™ Purification Instrument
iPrep™ gDNA Blood and Tissue Card, version 2
Blood samples (see below)
Blood Samples
The iPrep™ GeneCatcher™ gDNA Blood Kit is designed to purify high yield gDNA from various human blood samples including:
Fresh, whole blood
Blood collected in the presence of anti-coagulants such as EDTA or citrate.
Frozen blood samples or blood samples exposed to repeated freeze-thaw cycles
Old, archived blood samples and degraded samples
Sample Volume
The iPrep™ GeneCatcher™ gDNA Protocol is designed to purify gDNA from 350 μl human blood samples.
If sample volume is limited, you need use at least 200 μl sample volume. Do not use less than 200 μl blood sample volume as using less sample volume results in excessive bubble formation during the purification protocol thereby lowering the gDNA yield.
Follow the recommendations below to obtain the best results:
Maintain a sterile environment when handling DNA to avoid any contamination from DNases
Ensure that no DNases are introduced into the sterile solutions of the kit
Do not freeze the beads as this irreparably damages them. Store the beads at room temperature.
When using beads from the Reaction Cartridges, collect any solution from the foil by tapping the cartridge to deposit the solution at the bottom of the tube. Do not allow the beads to dry out as this renders them non-functional.
Discard Reaction Cartridges, iPrep™ Tips, and iPrep™ Tip Holders after use. Do not reuse.
Safety Information
Follow the safety guidelines below when using the iPrep™ GeneCatcher™ gDNA Blood Kit.
Treat all reagents supplied in the kit as potential irritants.
Always wear a suitable lab coat, disposable gloves, and protective goggles when handling whole blood samples.
Dispose of blood samples as biohazardous waste.
Isolating gDNA from Human Blood
Introduction
Instructions to isolate genomic DNA from human blood samples using the iPrep™ GeneCatcher™ gDNA Blood Kit with the iPrep™ Purification Instrument are described below.
Starting Material
Use this procedure to isolate genomic DNA from 350 μl human blood samples. See page 8 for sample volume.
Materials Needed
You will need the following materials:
Blood samples
iPrep™ Purification Instrument
iPrep™ gDNA Blood and Tissue Card, version 2.0 Components Supplied with the Kit
iPrep™ gDNA Blood Cartridge Kit
iPrep™ Sample and Elution Tubes
iPrep™ Tips
iPrep™ Tip Holders
Before Starting
Perform the following before starting:
Thaw frozen blood samples or mix the fresh blood samples and store on ice until use
Ensure that you have the iPrep™ gDNA Blood and Tissue Card, version 2.0 to run the tissue protocol
Make sure the iPrep™ Purification Instrument is unpacked and installed
Purification Protocol
Purify genomic DNA from human blood samples using the iPrep™ Purification Instrument as described below.
For details on using the iPrep™ Purification Instrument, refer to the manual supplied with the instrument.
Insert the iPrep™ gDNA Blood and Tissue Card, version 2.0 (available separately from Invitrogen) prior to turning on the instrument.
Ensure the power switch on the iPrep™ Instrument is on the OFF position.
Open the iPrep™ Card Slot and insert the iPrep™ gDNA Blood and Tissue Card into the slot in the correct orientation (arrow on the card is at the top and card label is facing your left side).
Using the power switch located on the left side of the instrument, turn ON the instrument. If the card is fully inserted in the correct orientation, all axes return to their original positions automatically. The digital display show the version for the iPrep™ which changes in few seconds to display the Main menu.
Press Start to run a protocol.
Press 2 to select the Blood protocol.
Open the iPrep™ door and remove the iPrep™ Cartridge Rack and iPrep™ Tip and Tube Rack to set up the platform.
Remove the desired number of iPrep™ gDNA Blood Cartridges from the box. To collect any solution from the foil, tap the cartridge to deposit the solution at the bottom of the tube. Note: You can load 1-13 cartridges on the rack depending on the number of samples that you wish to process. If you are loading less than 13 cartridges, ensure that the remaining plastic ware (tips and tubes) are also loaded in the same order as the cartridges
Insert one iPrep™ Sample and Elution Tube in position 11 (position with a hole) for each of the iPrep™ gDNA Blood Cartridge that is used.
Load the cartridges on the iPrep™ Cartridge Rack and insert the loaded rack on the iPrep™ platform.
Load the iPrep™ Tip and Tube Rack as follows:
l Load the first row (labeled as E) with 1-13 elution tubes without caps
l Keep the second row (labeled as T1) empty
l Load the third row (labeled as T2) with iPrep™ Tips in the iPrep™ Tip Holders
l Load the fourth row (labeled as S) with sample tubes without caps and add 350 μl blood sample to the sample tubes
Read the sample and elution tube barcode, if needed.
Insert the iPrep Tip and Tube rack on the iPrep™ platform as shown above.
Close the iPrep™ Door.
Press 1 to continue.
Select the appropriate elution volume on the display.
Ensure that you have loaded the cartridges, tubes, and tips in the appropriate positions, and sample and elution tubes do not have any caps. Make sure you have loaded a tube in position 11 of the cartridge.
Press Start. The automated purification protocol begins and various steps of the protocol including the approximate time remaining are displayed on the digital display.
Important: Do not open the door once the protocol has begun. To pause the protocol, press the Stop key. To resume the protocol after a pause, press the Start key. To cancel/stop the protocol, press the Stop key twice. For details, see the iPrep™ Instrument manual.
At the end of the run, the instrument beeps briefly and the digital display shows Protocol Finished for 10 seconds. The Main menu appears after 10 seconds.
Open the instrument door. Remove and cap the elution tubes containing the purified nucleic acid. Store the purified gDNA as described below.
Discard the used cartridges, tips, and sample tubes into biohazard waste. Do not reuse the cartridges.
To purify more samples using the same iPrep™ Card, load the racks with new cartridges, tips, and samples, and start the protocol as described above.
If you are not using the instrument, close the instrument door and turn the power switch to OFF.
Remove the iPrep™ gDNA Blood and Tissue Card and store the card in the box, protected from light.
Storing DNA
Use the purified DNA immediately for the desired downstream application.
Aliquot purified DNA and store at 4°C (short-term) or -20°C (long-term). Avoid repeated freezing and thawing.
DNA Quantitation and Analysis
DNA Yield
Perform DNA quantitation using UV absorbance at 260 nm or Quant-iT™ Kits.
UV Absorbance
Prepare a dilution of the DNA solution in 10 mM Tris-HCl, pH 7.5. Mix well. Measure the absorbance at 260 nm (A260) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against 10 mM Tris-HCl pH 7.5.
Calculate the concentration of DNA using the formula:
DNA (μg/ml) = A260 × 50 x dilution factor
For DNA, A260 = 1 for a 50 μg/ml solution measured in a cuvette with an optical path length of 1 cm.
Quant-iT™ Kits
The Quant-iT™ Kits provide a rapid, sensitive, and specific fluorescent method for dsDNA quantitation. The kit contains a state-of-the-art quantitation reagent, DNA standards for standard curve, and a pre-made buffer to allow fluorescent DNA quantitation using standard fluorescent microplate readers/fluorometers or the Qubit™ Fluorometer.
Analyzing DNA Quality
Typically, DNA isolated using the iPrep™ GeneCatcher™ gDNA Blood Kit has an A260/A280 >1.70 when samples are diluted in Tris-HCl (pH 7.5) indicating that the DNA is reasonably clean of proteins that could interfere with downstream applications. Purified gDNA may be analyzed by agarose gel electrophoresis to check the DNA quality (usually a single band at >40 kb with no smearing) and confirm the absence of contaminating RNA.
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