AGENCOURT ORAPURE Buccal Cell DNA Isolation Kit

Transcription

1 Buccal Cell DNA Isolation Kit Page 1 of 12 Please refer to for updated protocols and refer to MSDS instructions when handling or shipping any chemical hazards. AGENCOURT ORAPURE is a trademark of Agencourt Bioscience and is for laboratory use only. Agencourt Orapure: Table of Contents Introduction...1 Warnings and Precautions...1 Materials Supplied in the Buccal Cell Kit:...4 Swab Procedure:...5 Mouthwash Procedure:...8 Considerations for Increasing Yield:...11 Introduction The Agencourt Orapure Buccal Cell DNA Isolation Kit utilizes Agencourt s patented SPRI paramagnetic bead technology to isolate genomic DNA from buccal cells. The protocol can be performed in both 96-well and single tube formats. Buccal cells can be isolated using a standard swab technique or directly from mouthwash. Purification begins by resuspending buccal cells in a resuspension buffer followed by the addition of a lysis buffer. Immobilization of genomic DNA onto the magnetic particles occurs with the addition of PUR3, allowing the total genomic DNA to be separated away from contaminants using a magnetic field. Contaminants are rinsed away using a simple washing procedure, leaving the genomic DNA ready for elution from the magnetic particles. The 96-well plate format procedure is highly amenable to automation since it utilizes magnetic separation, thus eliminating the need for vacuum filtration or centrifugation. Genomic DNA from the Agencourt Orapure Buccal Cell Kit can be used in: Agarose gel analysis PCR 1 amplification Restriction enzyme digestion Human identity testing Membrane hybridizations (e.g., Southern and dot/slot blots). AFLP, RFLP, RAPD, microsatellite and SNP analyses (for genotyping, fingerprinting, etc.) Warnings and Precautions

2 Page 2 of Agencourt Bioscience Corp. kits are intended For Research Use only. 2. The U.S. Centers for Disease Control, the Food and Drug Administration, and the American Hospital Association recommend applying universal precautions when handling blood and body fluids to protect health care and laboratory workers. Under universal precautions, blood and body fluids are considered potentially infectious for blood-borne pathogens. It is recommended that workers protect themselves from contact with the fluids by using suitable barrier protection which includes gloves. Purified DNA may contain blood-borne pathogens, so effective barrier protections should be used throughout all stages of this procedure. Process Overview: Agencourt Orapure Buccal Cell Purification: 1. Resuspend buccal cells in R1 starting from either a mouthwash or swab sample 2. Lyse buccal cells in L2 3. Bind genomic DNA to paramagnetic bead particles using PUR3 4. Separate beads from contaminants 5. Wash the magnetic beads with ethanol to remove contaminants 6. Elute genomic DNA from magnetic particles

3 Page 3 of Transfer to new plate

4 Page 4 of 12 Materials Supplied in the Buccal Cell Kit: Agencourt Orapure Buccal Cell kit contains enough reagents to process 100 samples using the standard swab or mouthwash protocols. The reagents have a shelf life of 6 months if stored as directed. Reagent Description Storage Conditions Agencourt Orapure Buccal R1 Resuspension buffer 4 o C Agencourt Orapure Buccal L2 Lysis solution Room Temperature Agencourt Orapure Buccal PUR3 Binding solution (magnetic) 4 o C DO NOT FREEZE Agencourt Orapure Buccal E4 Elution buffer Room Temperature Kit contains 100 cytobrushes for the swab protocol. These are to be kept wrapped to ensure sterile conditions. Consumables and Hardware: Magnetic Separator: For 96 well format: SPRIPlate 96R ring magnetic plate [Agencourt #000219; For single tube format: SPRIStand magnetic tube rack [APN #001139; Reaction Plate: For 96 well format: 96 well 300µL round bottom microtiter plate [96 well 300µL round bottom microtiter plate [Costar # ; ] For single tube format: 1.7 ml microcentrifuge tubes, (ABGene #T5050G; Reagents: 70% Ethanol (Note: 70% ethanol is hygroscopic. Fresh 70% ethanol should be prepared for optimal results) Scope Mouthwash, 1998 Proctor & Gamble. Original Mint, Smooth Mint, or Peppermint.

5 Page 5 of 12 Swab Procedure: Important: This protocol is optimized for use with the included sterile cytobrushes only. The use of other types of swabs or brushes may diminish yield, and is not supported by Agencourt Bioscience. Subject should not eat or drink for 1 hour prior to collecting the buccal cell sample. 1. Add 200µl of R1 resuspension buffer to a clean 1.7ml microcentrifuge tube. 2. Unwrap a sterile cytobrush and swab the inside of one cheek a total of 20 times. Using an up and down motion, swab one cheek 10 times on one side of the brush then swab the same cheek 10 times with the other side of the brush. 3. Place the cytobrush (swab) in the microcentrifuge tube containing 200µl R1 resuspension buffer. Rotate the swab 10 times (spin between fingers) to release cells from the swab into the buffer. 4. With the swab still in the tube, add 300µl of L2 lysis solution. Gently rotate the swab 10 times to mix. The liquid can be pipetted directly onto the swab while in the tube. L2 ruptures the cellular membranes and releases the DNA. 5. Remove the swab from the tube, gently pressing it against the side of the tube to release as much liquid as possible from the fibers. Swab Protocol Summary: 1. No food or drink 1 hour before sample collection. 2. Add 200µl of R1 to a 1.7ml tube. 3. Swab 20 times with a sterile cytobrush. 4. Place the swab in the tube containing 200µl R1. Rotate swab 10 times to release cells. 5. Add 300µl of L2 lysis solution. Rotate the swab 10 times to mix. 6. Remove swab. Press it against the tube to release liquid from the fibers. 7. Incubate 15 minutes at room temperature. 8. Transfer 150µl lysate to a clean well or new microcentrifuge tube. 9. Add 150µl PUR3. Tipmix 10 times. Place on magnet 10 minutes to separate. 10. Aspirate and discard supernatant. 11. For a total of 3 washes: 12. Add 200µl of 70% ethanol, wait 30 seconds, remove supernatant and discard. 13. Dry at room temperature 2-5 minutes. 14. Add 40µl of E4. Tipmix 10 times to elute. 15. Place back on magnet 5-10 minute, then transfer eluant to a clean well or new microcentrifuge tube.

6 Page 6 of 12 The used swab can now be discarded. 6. Incubate samples at room temperature for 15 minutes. Incubation allows for complete lysis and release of DNA. 7. Transfer 150µl of the lysate to one well of a 96-well round bottom plate or to a clean 1.7mL microcentrifuge tube. Aspirate gently (20µl/sec) to prevent shearing of the genomic DNA. Repeat this transfer for any other swabs being purified. It is a good idea to keep the extra lysate - a second 150µl portion could be used if the experiment needs to be repeated, or multiple 150µl replicates could be processed in parallel to isolate extra genomic DNA in a single experiment. Higher yields can be obtained by purifying greater volumes of the lysate in 1.7mL microcentrifuge tubes. See the instructions at the end of the protocol for how to scale up the prep volume. 8. Add 150µl PUR3 binding solution to each well or tube containing 150µl lysate. Pipette tipmix 10 times until the mixture is homogeneous. Place sample plates/tubes on SPRIPlates/SPRIStand for 10 minutes to separate. PUR3 contains magnetic beads in a genomic DNA binding buffer. Shake the bottle to ensure that all the beads are in suspension before use. Once added, the solution should be pipette tipmixed 10 times or until it appears homogeneous. After magnetic separation, the supernatant should be clear and the beads should form a ring near the bottom of the plate or a tight mass on the side of the tube before proceeding to the next step. 9. Slowly aspirate and discard the clear supernatant from the plate or tube while it is situated on the magnet. You should be able to move the tips to the bottom of the well/tube in the center without disturbing the beads. If the magnetic beads become agitated, more magnetic separation time may be required before attempting the supernatant removal step. The discarded supernatant should be free of magnetic beads. 10. Dispense 200µl of 70% ethanol into each well or tube. Wait 30 seconds, then remove the ethanol wash solution and discard. Repeat for a total of three washes. The plate must be left on the magnet during the ethanol washes. At this point, the beads have the genomic DNA bound to them. Be sure to remove as much of the ethanol as possible, as it may contain residual contaminants. Ethanol is hydroscopic; be sure to use fresh 70% ethanol for best results. If the concentration of the ethanol is less than 70%, some of the product may be rinsed away.

7 Page 7 of Briefly dry the plate at room temperature for 2-5 minutes. Overdrying the DNA onto the beads will make it difficult to elute the genomic DNA and may result in decreased yield. Do not use a SpeedVac to dry the samples. 12. Add 40µl of E4 elution buffer to each sample to elute. For plates pipette tipmix 10 times; for tubes flick/tap the tube with a finger several times to complete elution. E4 elution buffer contains RNase. As alternatives, DiH2O or 10mM Tris-Cl, ph8.5 could be used for elution. Genomic DNA may denature slightly in DiH2O. It is not necessary for the beads to go back into solution for complete elution to occur. 13. Place the plate or tubes back on the magnet and wait 5-10 minutes for the beads to separate. Transfer the clear eluant to clean well/ plate or a fresh microcentrifuge tube. The beads may separate in 1-2 minutes, but it is recommended to wait 5-10 minutes to allow the beads to compact more. This will make the transfer easier to perform. Transfer cleared eluant to a clean plate or tube for use in downstream reactions. The original plate/tube must be left on the magnet during the transfer to minimize bead carry-over. The DNA should be stored at -20 C.

8 Page 8 of 12 Mouthwash Procedure: Important: This protocol is optimized for use with Scope Brand mouthwash only. Other mouthwashes may diminish yield and are not supported by Agencourt Bioscience. Subject should not eat or drink for 1 hour prior to collecting the buccal sample. 1. Give subject 10 ml of mouthwash. Swish vigorously for 20 seconds from cheek to cheek - DO NOT GARGLE. Spit mouthwash into a 50 ml conical tube. When swishing, rinse cheeks with mouthwash from one side to the other without gargling. Swishing must be done for at least 20 seconds. This will maximize buccal cell extraction. Gargling introduces phlegm from throat which is not conducive to resuspension or automated tipmixing. When spitting, it is important to get the entire mouthwash sample into the tube to maximize yield. Close the tube tightly to minimize exposure to air, especially if samples must be stored long term prior to purification. Mouthwash samples will produce the most intact genomic DNA if processed within 3 hours of collecting the sample. A good percentage of intact genomic DNA can still be isolated within 3 days with storage of the mouthwash at room temperature. Between 3-10 days the DNA in the mouthwash will continue to degrade, but is still usable for PCR. After 10 days, the DNA will be too degraded for PCR. Mouthwash Protocol Summary: 1. No food or drink 1 hour before sample collection. 2. Swish with 10mL mouthwash for 20 seconds. Spit into conical tube. 3. Centrifuge 3000 x g for 10 minutes to pellet cells. 4. Discard supernatant by decanting. 5. Resuspend pellet in 400 µl R1. 6. Add 600 µl L2 and gently tipmix. 7. Incubate 30 minutes at room temperature. 8. Transfer 150 µl lysate to a clean well or new microcentrifuge tube. 9. Add 150 µl PUR3. Tipmix 10 times. Place on magnet 10 minutes to separate. 10. Aspirate and discard supernatant. 11. For a total of 3 washes: 12. Add 200 µl of 70% ethanol, wait 30 seconds, remove supernatant and discard. 13. Dry at room temperature 2-5 minutes. 14. Add 40 µl of E4. Tipmix 10 times to elute. 15. Place back on magnet 5-10 minute, then transfer eluant to a clean well or new microcentrifuge tube. 2. Spin tube at 3000 x g for 10 minutes to pellet cells from the mouthwash. The following are settings for two common centrifuges and rotors:

9 Page 9 of 12 Beckman GH 3.8 rotor with MicroPlus carrier: Jouan P60 rotor with microplate carrier: 3000 x g = 3625 rpm 3000 x g = 3170 rpm 3. Discard the clear supernatant by carefully decanting. When removing the supernatant, take care not to disturb the cell pellet as this may reduce yield. 4. Resuspend pellet in 400 µl R1 resuspension buffer and mix by vortexing or pipette tipmixing cells until a homogeneous solution is achieved. Vortexing (medium speed), orbital shaking (300 RPM), pipette tipmixing (50 µl/second), or a combination of these will accomplish resuspension of the pellet. The cell pellet should be completely resuspended so that the mixture appears homogeneous and has no cell clumps before continuing. 5. Add 600 µl L2 lysis solution and gently pipette tipmix. L2 ruptures the cellular membranes and releases the DNA. Vigorous pipette tipmixing is not recommended at this point as the genomic DNA may be sheared. 6. Incubate samples at room temperature for 30 minutes. Incubation allows for complete lysis of the large cell pellet. 7. Transfer 150 µl of the lysate to one well of a 96-well round bottom plate or to a clean 1.7 ml microcentrifuge tube. Aspirate gently (20 µl/sec) to prevent shearing of the genomic DNA. Repeat this transfer for any other mouthwash samples being purified. It is a good idea to keep the extra lysate - a second 150 µl portion could be used if the experiment needs to be repeated, or multiple 150 µl replicates could be processed in parallel to isolate extra genomic DNA in a single experiment. Higher yields can be obtained by purifying greater volumes of the lysate in 1.7 ml microcentrifuge tubes. See the instructions at the end of the protocol for how to scale up the prep volume. 8. Add 150 µl PUR3 binding solution to each well or tube containing 150 µl lysate. Pipette tipmix 10 times until the mixture is homogeneous. Place sample plates/tubes on SPRIPlates/SPRIStand for 10 minutes to separate. PUR3 contains magnetic beads in a genomic DNA binding buffer. Shake the bottle to ensure that all the beads are in suspension before use. Once added, the solution should be pipette tipmixed 10 times or until it appears homogeneous. After magnetic separation, the

10 Page 10 of 12 supernatant should be clear and may have the same color as the Scope mouthwash. The beads should form a ring near the bottom of the plate or a tight mass on the side of the tube before proceeding to the next step. 9. Slowly aspirate and discard the clear supernatant from the plate or tube while it is situated on the magnet. You should be able to move the tips to the bottom of the well/tube in the center without disturbing the beads. If the magnetic beads become agitated, more magnetic separation time may be required before attempting the supernatant removal step. The discarded supernatant should be free of magnetic beads. 10. Dispense 200 µl of 70% ethanol into each well or tube. Wait 30 seconds, then remove the ethanol wash solution and discard. Repeat for a total of three washes. The plate must be left on the magnet during the ethanol washes. At this point, the beads have the genomic DNA bound to them. Be sure to remove as much of the ethanol as possible, as it may contain residual contaminants. Ethanol is hydroscopic; be sure to use fresh 70% ethanol for best results. If the concentration of the ethanol is less than 70%, some of the product may be rinsed away. 11. Briefly dry the plate at room temperature for 2-5 minutes. Overdrying the DNA onto the beads will make it difficult to elute the genomic DNA and may result in decreased yield. Do not use a SpeedVac to dry the samples. 12. Add 40 µl of E4 elution buffer to each sample to elute. For plates pipette tipmix 10 times; for tubes flick/tap the tube with a finger several times to complete elution. E4 elution buffer contains RNase. As alternatives, DiH 2 O or 10mM Tris-Cl, ph8.5 could be used for elution. Genomic DNA may denature slightly in DiH 2 O. It is not necessary for the beads to go back into solution for complete elution to occur. 13. Place the plate or tubes back on the magnet and wait 5-10 minutes for the beads to separate. Transfer the clear eluant to clean well/ plate or a fresh microcentrifuge tube. The beads may separate in 1-2 minutes, but it is recommended to wait 5-10 minutes to allow the beads to compact more. This will make the transfer easier to perform. Transfer cleared eluant to a clean plate or tube for use in downstream reactions. The original plate/tube must be left on the magnet during the transfer to minimize bead carry-over. The DNA should be stored at -20 C.

11 Page 11 of 12 Considerations for Increasing Yield: To increase the yield of the Agencourt Orapure Buccal Cell kit, increase the total volume of lysate purified with PUR3 binding solution (up to 500 µl). These larger volumes must be done in 1.7 ml microcentrifuge tubes and the subsequent reagent volumes (PUR3, 70% Ethanol and E4 elution buffer) will need to be scaled linearly with respect to the input transfer volume. Slightly longer magnetic separation times may be required to fully clear the larger samples. The mouthwash based buccal cell collection technique harvests more cells than the cytobrush swab. To get the maximum yield possible with the Agencourt Orapure Buccal Cell kit, Agencourt suggests purifying 500 µl of standard mouthwash lysate. Purification of 500µl swab lysate will also give a substantial increase in yield. The Agencourt Orapure Buccal Cell kit was designed to purify 100 samples based on the 150µl transfer protocols. Using larger transfer volumes the kit will purify the following numbers of samples: Transfer Volume Number of Purifications 150 µl µl µl µl µl Follow the standard protocols for processing swabs or mouthwash though incubation of the samples in L2 buffer. Once the incubation is complete transfer the desired amount of lysate (up to 500 µl) to a clean 1.7 ml microcentrifuge tube. 2. Add PUR3 binding solution to each sample according to the following equation: Volume PUR3 = 1.0 µl x Transfer Volume Pipette tipmix 10 times until the mixture is homogeneous. Place sample tubes on a SPRIStand for minutes to separate. The solution should be clear before continuing. PUR3 contains magnetic beads in a genomic DNA binding buffer. Shake the bottle to ensure that all the beads are in suspension before use. Once added, the solution should be pipette tipmixed 10 times or until it appears homogeneous. The beads should form a tight mass on the side of the tube before proceeding to the next step. 3. Slowly aspirate and discard the clear supernatant from tube while it is situated on the magnet.

12 Page 12 of 12 You should be able to move the tips to the bottom of the tube without disturbing the beads. If the magnetic beads become agitated, more magnetic separation time may be required before attempting the supernatant removal step. The discarded supernatant should be free of magnetic beads. 4. Dispense 70% ethanol into each tube according to the following equation: Volume 70% Ethanol = 2.0 µl x Transfer Volume Wait 30 seconds, then remove the ethanol wash solution and discard. Repeat for a total of three washes. The plate must be left on the magnet during the ethanol washes. At this point, the beads have the genomic DNA bound to them. Be sure to remove as much of the ethanol as possible, as it may contain residual contaminants. Ethanol is hydroscopic; be sure to use fresh 70% ethanol for best results. If the concentration of the ethanol is less than 70%, some of the product may be rinsed away. 5. Briefly dry the tubes at room temperature for 5 minutes. Overdrying the DNA onto the beads will make it difficult to elute the genomic DNA and may result in decreased yield. Do not use a SpeedVac to dry the samples. 6. Add E4 elution buffer to each sample according to the following equation: Volume E4 = 0.27 µl x Transfer Volume Pipette tipmix 10 times or flick/tap the tube with a finger several times to complete elution. E4 elution buffer contains RNase. As alternatives, DiH2O or 10mM Tris-Cl, ph8.5 could be used for elution. Genomic DNA may denature slightly in DiH2O. It is not necessary for the beads to go back into solution for complete elution to occur. 7. Place the tubes back on the magnet and wait 5-10 minutes for the beads to separate. Transfer the clear eluant to clean plate or a fresh microcentrifuge tube. The beads may separate in 1-2 minutes, but it is recommended to wait 5-10 minutes to allow the beads to compact more. This will make the transfer easier to perform. Transfer cleared eluant to a clean tube for use in downstream reactions. The original tube must be left on the magnet during the transfer to minimize bead carry-over. The DNA should be stored at -20 C. LIMITED USE LABEL LICENSE This product is covered by at least one or more claims of US patents Nos. 5,898,071, 5,705,628, and/or 6,534,262, which are exclusively licensed to Agencourt Bioscience Corporation. This product is sold strictly for the use of the buyer and the buyer is not authorized to transfer this product [or any materials made using this product] to any third party. 1 The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-La Roche, Ltd.