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Affymetrix Genome-wide Human SNP Assay
Affymetrix全基因组人单核苷酸多态性试验   

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Abstract

To assess genomic variation, it is possible to identify the single nucleotide polymorphisms (SNP) which an individual carries. Using the Affymetrix Genome-wide Human SNP Assay, it is possible to assess 906,600 SNPs on a single array. This protocol, the next iteration of the GeneChip Mapping 500K array set, is based directly on the manufacturers’ protocol and shows steps which are highly similar to that which is found here: http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf.

Keywords: Single nucleotide polymorphisms(单核苷酸多态性), Array(阵列), Human cells(人体细胞)

Materials and Reagents

  1. 96-well V-bottom plates, elution catch (Greiner Bio-One GmbH, catalog number: 651101 )
  2. Greiner UV star transparent, 96-well (Greiner Bio-One GmbH, catalog number: 82050-788 )
    Note: Currently, it is “VWR International, catalog number: 82050-788”.
  3. Multiplate 96-well unskirted PCR plate (Bio-Rad Laboratories, catalog number: MLP-9601 )
  4. Multiscreen deep well plate (Merck Millipore Corporation, catalog number: MDRLN0410 )
  5. 2.4 ml deep well storage plate/pooling plate (Greiner Bio-One GmbH, catalog number: 780280 )
  6. 100 ml solution basin (VWR International, LabcorTM, catalog number: 730-014 )
  7. 55 ml solution basin (VWR International, LabcorTM, catalog number: 730-004 )
  8. 15 ml centrifuge tubes (VWR International, catalog number: 20171-020 )
  9. 50 ml centrifuge tubes (VWR International, catalog number: 21008-178 )
  10. 2.0 ml Eppendorf tubes (VWR International, catalog number: 20901-540 )
  11. 0.2 ml 12-strip tubes (VWR International, catalog number: 21008-940 )
  12. Lab tape
  13. Tough-Spots®
  14. Microseal ‘B’ adhesive seal (Bio-Rad Laboratories, model: MSB1001 )
  15. MicroAmp® clear adhesive film applied (Biosystems, catalog number: 4306311 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4306311”.
  16. Kimwipes (source as desired)
  17. Pipet tips as appropriate for pipettes (source as desired)
  18. AccuGENE® water, molecular biology-grade (Affymetrix, catalog number: 71786 )
  19. GenElute Mammalian Genomic DNA Miniprep (Sigma-Aldrich, catalog number: G1N70 )
  20. Affymetrix SNP 6 Core Reagent Kit, 100 reactions (Affymetrix, catalog number: 901706 )
    1. 10x Sty I buffer/NE buffer 3 (New England Biolabs)
    2. T4 DNA Ligase (400 U/μl) (New England Biolabs)
    3. T4 DNA Ligase Buffer (10x) (New England Biolabs)
    4. Adaptor, sty (50 μM)
    5. PCR Primer 002 (100 μM)
    6. 10x Nsp I buffer/NE buffer 2 (New England Biolabs)
    7. NspI (10 U/μl) (New England Biolabs)
    8. Adaptor, nsp (50 μM)
    9. Elution buffer (Buffer EB)
    10. Fragmentation buffer (10x)
    11. Fragmentation reagent (DNase I)
    12. DNA Labeling reagent
    13. Terminal deoxynucleotidyl transferase (TdT) (30 U/μl)
    14. Terminal deoxynucleotidyl transferase buffer (TdT buffer) (5x)
    15. Oligo control reagent (OCR), 0100
    16. 10 mg/ml BSA (100x)
    17. Hybridization Master Mix
  21. DNA marker (Bionexus, catalog number: BN2050 )
  22. 2% TBE gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: G8008-02 )
  23. Gel loading solution (Faster Better Media, catalog number: SB5N-8 )
  24. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  25. 50 ml magnetic beads (AMPure, catalog number: A63881 )
    Note: It is named “Agencourt AMPure XP-PCR Purification” on Beckman Coulter website.
  26. 4% TBE gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: G8008-04 )
  27. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
  28. MES sodium salt (Sigma-Aldrich, catalog number: M3885 )
    Note: All catalog numbers are listed in the Affymetrix datasheet (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf).
  29. Clontech TITANIUMTM DNA Amplification Kit (Clontech, catalog number: 639240 ) (see Recipes)
  30. 12x MES Stock Solution (see Recipes)

Equipment

  1. Applied Biosystems units
    1. 2720 Thermal Cycler
    2. GeneAmp® PCR System 9700
    3. GeneAmp® PCR System 9700 (silver block or gold-plated silver block)
  2. 48 Genome-Wide Human SNP Array 6.0 (one array per sample)
  3. GeneChip® Hybridization Oven 640
  4. Jitterbug
  5. Spectrophotometer plate reader
  6. Vacuum Manifold (Merck Millipore Corporation)
  7. Vortexer
  8. Cooler (chilled to -20 °C)
  9. Cooling chamber (chilled to 4 °C) (do not freeze)
  10. Cooling chamber (double, chilled to 4 °C) (do not freeze)
  11. Ice bucket, filled with ice
  12. Marker, fine point, permanent
  13. Microcentrifuge
  14. Pipet, 12-channel P1200
  15. Pipet, 12-channel P200
  16. Pipet, 12-channel P20 (accurate to within ± 5%)
  17. Pipet, serological
  18. Pipet, single channel P1000
  19. Pipet, single channel P200
  20. Plate centrifuge with deep-well capacity (54 mmH x 160 g)
  21. Optical plates
  22. Centrifuge plate holders
    Notes:
    1. Use only the PCR plate, adhesive film and thermal cyclers listed here.
    2. All relevant equipment numbers are listed in the Affymetrix datasheet.
    (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf)

Procedure

Genomic DNA Plate Preparation

  1. Extract DNA from your cells of interest using an extraction protocol which yields high quality, contaminant-free DNA free from inhibitors (e.g., GenElute Mammalian Genomic DNA Miniprep).
    Note: DNA should be around 1.8 for 260/280 and 2.0 for 260/230 ratios when examining the optical density using a spectrophotometer.
  2. Thoroughly mix the genomic DNA by vortexing at high speed for 3 sec.
  3. Determine the concentration of each sample (e.g., Nanodrop).
  4. Dilute each sample to 50 ng/μl using reduced EDTA TE buffer.
  5. Thoroughly mix the diluted DNA by vortexing at high speed for 3 sec.
  6. Vortex the plate of genomic DNA at high speed for 10 sec, then spin down at 560 x g for 30 sec.
  7. Aliquot 5 μl of each DNA to the corresponding wells of two 96-well reaction plates. 5 μl of the 50 ng/μl working stock is equivalent to 250 ng genomic DNA per well. Two replicates of each sample are required for this protocol: One for Nsp and one for processing Sty.
  8. Seal each plate with adhesive film.

Stage 1: Sty Restriction Enzyme Digestion
During this stage, the genomic DNA is digested by the StyI restriction enzyme.

  1. Thaw/place the following reagents on ice:
    1. NE Buffer 3
    2. BSA
    3. Genomic DNA (if frozen)
    4. AccuGENE water
  2. Label the following tubes, then place in the cooling chamber:
    1. One strip of 12 tubes labeled “Dig”.
    2. One 2.0 ml Eppendorf tube labeled “Dig MM”.
  3. Prepare the plate with genomic DNA as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Place back in the cooling chamber on ice.
  4. Prepare the reagents (except for the enzyme) as follows:
    1. Vortex 3 times, 1 sec each time.
    2. Pulse spin for 3 sec.
    3. Place in the cooling chamber.
  5. Preheat the Thermal Cycler lid, however leave the block at room temperature.
    Note: Leave the STYI enzyme at -20 °C until ready to use.
  6. Prepare the Sty digestion Master Mix by adding the volumes of the following reagents to the 2.0 ml Eppendorf tube as in Table 1:
    1. AccuGENE water
    2. NE Buffer 3
    3. BSA
  7. Remove the StyI enzyme from the freezer and immediately place in a cooler.
  8. Pulse spin the enzyme for 3 sec.
  9. Immediately add the enzyme to the master mix, then place remaining enzyme back in the cooler.
  10. Vortex the master mix at high speed 3 times, 1 sec each time.
  11. Pulse spin for 3 sec.
  12. Place in the cooling chamber.
  13. Return any remaining enzyme to the freezer.
  14. Proceed immediately to Add Sty Digestion Master Mix to Samples.

    Table 1. StyI Digestion Master Mix
    Reagent
    1 Sample
    48 Samples (15% Extra)
    AccuGENE® water
    11.55 μl
    637.6 μl
    NE Buffer 3 (10x)
    2 μl
    110.4 μl
    BSA (100x; 10 mg/ml)
    0.2 μl
    11 μl
    StyI (10 U/μl)
    1 μl
    55.2 μl
    Total
    14.75 μl
    814.2 μl

    Add Sty Digestion Master Mix to Samples (stage 1, steps 15-22)
  15. To add the Sty Digestion Master Mix to samples, use a single channel P200 pipet, aliquot 67 μl of Sty Digestion Master Mix to each tube of the strip tubes labeled Dig.
  16. Using a 12-channel P20 pipet, add 14.75 μl of Sty Digestion Master Mix to each DNA sample in the cooling chamber on ice. The total volume in each well is now 19.75 μl.
    Reagent
    Volume
    Genomic DNA (50 ng/μl)
    5 μl
    Digestion Master Mix
    14.75 μl
    Total Volume
    19.75 μl
  17. Seal the plate tightly with adhesive film.
  18. Vortex the center of the plate at high speed for 3 sec.
  19. Spin down the plate at 560 x g for 30 sec.
  20. Ensure that the lid of thermal cycler is preheated.
  21. Load the plate onto the thermal cycler and run the GW5.0/6.0 Digest program.
    GW5.0/6.0 Digest Program
    Temperature
    Time
    37 °C
    120 min
    65 °C
    20 min
    4 °C
    Hold
  22. When the program is finished, remove the plate and spin it down at 560 x g for 30 sec.

Stage 2: Sty Ligation
During this stage, the digested samples are ligated using the Sty Adaptor.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. Adaptor StyI
    2. T4 DNA Ligase Buffer (10x)
    3. Sty digested samples (if frozen)
    4. AccuGENE water
    Note: Leave the T4 DNA Ligase at -20 °C until ready to use.
  2. Label the following tubes, then place in the cooling chamber:
    1. One strip of 12 tubes labeled “Lig”
    2. A 2.0 ml Eppendorf tube labeled “Lig MM”
    3. Solution basin
  3. Prepare the digested samples as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Place back in the cooling chamber on ice.
  4. To prepare the reagents:
    1. Vortex at high speed 3 times, 1 sec each time (except for the enzyme).
    2. Pulse spin for 3 sec.
    3. Place in the cooling chamber.
  5. Preheat the Thermal Cycler lid, however leave the block at room temperature. The lid must be preheated before samples are loaded.
    Note: T4 DNA Ligase Buffer (10x) contains ATP and should be thawed on ice. Vortex the buffer as long as necessary before use to ensure precipitate is re-suspended and that the buffer is clear. Avoid multiple freeze-thaw cycles per vendor instructions.

    Prepare the Sty Ligation Master Mix (stage 2, steps 6-13)
  6. Keeping all reagents and tubes on ice, prepare the Sty Ligation Master Mix by adding to the 2.0 ml Eppendorf tube the following reagents based on the volumes shown in Table 2:
    1. Adaptor StyI
    2. T4 DNA Ligase Buffer (10x)

    Table 2. StyI Ligation Master Mix
    Reagent
    1 Sample
    48 Samples (15% extra)
    T4 Ligase Buffer (10x)
    2.5 μl
    150 μl
    Adaptor StyI (50 μM)
    0.75 μl
    45 μl
    T4 DNA Ligase (400 U/μl)
    2 μl
    120 μl
    Total
    5.25 μl
    315 μl

  7. Remove the T4 DNA Ligase from the freezer and immediately place in the cooler on ice.
  8. Pulse spin the T4 DNA Ligase for 3 sec.
  9. Immediately add the T4 DNA Ligase to the master mix; then place back in the cooler.
  10. Vortex the master mix at high speed 3 times, 1 sec each time.
  11. Pulse spin for 3 sec.
  12. Place the master mix on ice.
  13. Proceed immediately to Add Sty Ligation Master Mix to Reactions.

    Add Sty Ligation Master Mix to Reactions (stage 2, steps 14-27)
  14. Add Sty Ligation Master Mix to samples using a single channel P100 pipet to aliquot 25 μl of Sty Ligation Master Mix to each tube of the strip tubes on ice.
  15. Using a 12-channel P20 pipet, aliquot 5.25 μl of Sty Ligation Master Mix to each reaction on the Sty Digestion Stage Plate. The total volume in each well is now 25 μl.
    Reagent
    Volume
    Sty Digested DNA
    19.75 μl
    Sty Ligation Master Mix
    5.25 μl
    Total
    25 μl
  16. Seal the plate tightly with adhesive film.
  17. Vortex the center of the plate at high speed for 3 sec.
  18. Spin down the plate at 560 x g for 30 sec.
  19. Ensure that the thermal cycler lid is preheated.
  20. Load the plate onto the thermal cycler and run the GW5.0/6.0 Ligate program.
    GW5.0/6.0 Ligate Program
    Temperature
    Time
    16 °C
    180 min
    70 °C
    20 min
    4 °C
    Hold
  21. When the GW5.0/6.0 Ligate program is finished, remove the plate and spin it down at 560 x g for 30 sec.
  22. Place the plate in a cooling chamber on ice.
  23. Dilute each reaction as follows:
    1. Pour 10 ml AccuGENE water into the solution basin.
    2. Using a 12-channel P200 pipet, add 75 μl of the water to each reaction.
    The total volume in each well is 100 μl.
    Reagent
    Volume
    Sty Ligated DNA
    25 μl
    AccuGENE water
    75 μl
    Total
    100 μl
  24. Seal the plate tightly with adhesive film.
  25. Vortex the center of the plate at high speed for 3 sec.
  26. Spin down the plate at 560 x g for 30 sec.
  27. Store the plate in a cooling chamber on ice for up to 60 min. If not proceeding directly to the next step, store the plate at -20 °C.
    Note: It is crucial to dilute the ligated DNA with AccuGENE water prior to PCR.

Stage 3: Sty PCR
During this stage you will transfer equal amounts of each Sty ligated sample into three fresh 96-well plates; prepare the Sty PCR Master Mix, and add it to each sample; place each plate on a thermal cycler and run the GW6.0 PCR program; and confirm the PCR by running 3 μl of each PCR product on a 2% TBE gel or an E-Gel® 96 2% agarose gel.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. TITANIUM Taq PCR Buffer
    2. dNTPs
    3. PCR Primer 002
    4. Sty ligated samples (if frozen)
    5. AccuGENE water
    6. GC-Melt
    7. Solution basin
    Note: Leave the TITANIUM Taq DNA Polymerase at -20 °C until ready to use.
  2. Label the following tubes, then place in the cooling chamber or on ice, as appropriate:
    1. Three 96-well reaction plates labeled “P1”, “P2”, “P3”
    2. One 50 ml Falcon tube labeled “PCR MM”
  3. Prepare the Sty ligated samples as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Label the plate Lig.
    4. Place back in the cooling chamber on ice.
  4. To prepare the reagents:
    1. Vortex at high speed 3 times, 1 sec each time (except for the enzyme).
    2. Pulse spin for 3 sec.
    3. Place in a cooling chamber.
  5. Preheat the Thermal Cycler lid, however leave the block at room temperature.

    Aliquot Sty Ligated DNA to the PCR Plates (stage 3, steps 6-7)
  6. Working one row at a time and using a 12-channel P20 pipet, transfer 10 μl of each Sty ligated sample to the corresponding well of each PCR plate.
  7. Seal each plate with adhesive film, and leave in cooling chambers on ice.

    Prepare the Sty PCR Master Mix (stage 3, steps 8-13)
  8. To prepare the Sty PCR Master Mix, keep the 50 ml Falcon tube in the cooling chamber and add the reagents as shown in Table 3 (except for the Taq DNA polymerase).

    Table 3. Sty PCR Master Mix for 48 Samples

    Reagent
    1 Reaction
    3 PCR Pates,
    48 Samples each plate
    (15% Extra)
    AccuGENE water
    39.5 μl
    6.541 ml
    TITANIUM Taq PCR Buffer (10x)
    10 μl
    1.656 ml
    GC-Melt (5 M)
    20 μl
    3.312 ml
    dNTP (2.5 mM each)
    14 μl
    2.318 ml
    PCR Primer 002 (100 μM)
    4.5 μl
    0.745 ml
    TITANIUM Taq DNA Polymerase (50x) (do not add until ready to aliquot master mix to ligated samples)
    2 μl
    0.331 ml
    Total
    90 μl
    14.903 ml

  9. Remove the TITANIUM Taq DNA Polymerase from the freezer and immediately place in a cooler.
  10. Pulse spin the Taq DNA polymerase for 3 sec.
  11. Immediately add the Taq DNA polymerase to the master mix; then return the tube to the cooler on ice.
  12. Vortex the master mix at high speed 3 times, 1 sec each time.
  13. Pour the mix into the solution basin, keeping the basin on ice.

    Add Sty PCR Master Mix to Samples (stage 3, steps 14-18)
  14. To add Sty PCR Master Mix to samples, use a 12-channel P200 pipet and add 90 μl Sty PCR Master Mix to each sample. To avoid contamination, change pipet tips after each dispense. The total volume in each well is 100 μl.
  15. Seal each reaction plate tightly with adhesive film.
  16. Vortex the center of each reaction plate at high speed for 3 sec.
  17. Spin down the plates at 560 x g for 30 sec.
  18. Keep the reaction plates in cooling chambers on ice until loaded onto the thermal cyclers.

    Load Sty PCR Plates onto Thermal Cyclers (stage 3, steps 19-22)
  19. Transfer the plates to the Main Lab.
  20. Ensure that the thermal cycler lids are preheated. The block should be at room temperature.
  21. Load each reaction plate onto a thermal cycler.
  22. Run the GW5.0/6.0 PCR program. The program varies depending upon the thermal cyclers you are using. The example used here is for GeneAmp® PCR System 9700, however another protocol can be found in the manufacturers’ instructions.
    GW5.0/6.0 PCR Program for GeneAmp® PCR System 9700
    Temperature
    Time
    Cycles
    94 °C
    3 min
    1x
    94 °C
    30 sec
    |
    60 °C
    45 sec
    30x
    68 °C
    15 sec
    |
    68 °C
    7 min
    1x
    4 °C
    Hold

    Volume:
    100 μl

    Specify Maximum mode.

    Running Gels (stage 3, steps 23-33)
    Note: The instructions below are for running 2% TBE gels. To ensure consistent results, take 3 μl aliquot from each PCR.
  23. When the GW5.0/6.0 PCR program is finished, remove each plate from the thermal cycler.
  24. Spin down plates at 560 x g for 30 sec.
  25. Place plates in cooling chambers on ice or keep at 4 °C.
  26. Label three fresh 96-well reaction plates P1Gel, P2Gel and P3Gel.
  27. Aliquot 3 μl of 2x Gel Loading Dye to each well in rows A through D of the fresh, labeled PXGel plates.
  28. Using a 12-channel P20 pipet, transfer 3 μl of each PCR product from the 3 Sty PCR plates to the corresponding plate, row and wells of the PXGel plates. Example: 3 μl of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel.
  29. Seal the PXGel plates.
  30. Vortex the center of each PXGel plate, then spin them down at 560 x g for 30 sec.
  31. Load the total volume from each well of each PXGel plate onto 2% TBE gels.
  32. Run the gels at 120 V for 40 min to 1 h.
  33. Verify that the PCR product distribution is between ~200 bp to 1,100 bp (example of gel below, Figure 1).


    Figure 1. Example of PCR products run on 2% TBE agarose gel at 120 V for 1 h. Average product distribution is between ~200 to 1,100 bp.

Stage 4: Nsp Restriction Enzyme Digestion
During this stage, the genomic DNA is digested by the Nsp I enzyme.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. NE Buffer 2
    2. BSA
    3. Genomic DNA (if frozen)
    4. AccuGENE water
  2. Label the following tubes, then place in the cooling chamber or on ice, as appropriate:
    1. One strip of 12 tubes labeled “Dig”
    2. A 2.0 ml Eppendorf tube labeled “Dig MM”
  3. Prepare the plate with genomic DNA as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Place back in the cooling chamber on ice.
  4. Prepare the reagents (except for the enzyme) as follows:
    1. Vortex 3 times, 1 sec each time.
    2. Pulse spin for 3 sec.
    3. Place in the cooling chamber.
  5. Preheat the Thermal Cycler lid, however leave the block at room temperature.
    Note: Leave the NSP I enzyme at -20 °C until ready to use.

    Prepare the Nsp Digestion Master Mix (stage 4, steps 6-13)
  6. Keeping all reagents and tubes on ice, prepare the Nsp Digestion Master Mix by adding the appropriate volumes of the following reagents from Table 4:
    1. AccuGENE water
    2. NE Buffer 2
    3. BSA
  7. Remove the Nsp I enzyme from the freezer and immediately place in a cooler.
  8. Pulse spin the enzyme for 3 sec.
  9. Immediately add the enzyme to the master mix, then place remaining enzyme back in the cooler.
  10. Vortex the master mix at high speed 3 times, 1 sec each time.
  11. Pulse spin for 3 sec.
  12. Place in the cooling chamber.
  13. Return any remaining enzyme to the freezer.

    Table 4. NspI Digestion Master Mix
    Reagent
    1 Sample
    48 Samples (15% Extra)
    AccuGENE® Water
    11.55 μl
    637.6 μl
    NE Buffer 2 (10x)
    2 μl
    110.4 μl
    BSA (100x; 10 mg/ml)
    0.2 μl
    11 μl
    NspI (10 U/μl)
    1 μl
    55.2 μl
    Total
    14.75 μl
    814.2 μl

    Add Nsp Digestion Master Mix to Samples (stage 4, steps 14-21)
  14. To add Nsp Digestion Master Mix to samples, use a single channel P200 pipet and aliquot 67 μl of Nsp Digestion Master Mix to each tube of the strip tubes labeled Dig.
  15. Using a 12-channel P20 pipet, add 14.75 μl of Nsp Digestion Master Mix to each DNA sample in the cooling chamber on ice. The total volume in each well is now 19.75 μl.
    Reagent
    Volume
    Genomic DNA (50 ng/μl)
    5 μl
    Nsp Digestion Master Mix
    14.75 μl
    Total Volume
    19.75 μl
  16. Seal the plate tightly with adhesive film.
  17. Vortex the center of the plate at high speed for 3 sec.
  18. Spin down the plate at 560 x g for 30 sec.
  19. Ensure that the lid of thermal cycler is preheated.
  20. Load the plate onto the thermal cycler and run the GW5.0/6.0 Digest program.
    GW5.0/6.0 Digest Program
    Temperature
    Time
    37 °C
    120 min
    65 °C
    20 min
    4 °C
    Hold
  21. When the program is finished, remove the plate and spin it down at 560 x g for 30 sec.

Stage 5: Nsp Ligation
During this stage, the digested samples are ligated using the Nsp Adaptor.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. Adaptor NspI
    2. T4 DNA Ligase Buffer (10x)
    3. Nsp digested samples (if frozen)
    4. AccuGENE water
  2. Label the following tubes, then place in the cooling chamber or on ice, as appropriate:
    1. One strip of 12 tubes labeled “Lig”
    2. A 2.0 ml Eppendorf tube labeled “Lig MM”
    3. Solution basin
  3. Prepare the digested samples as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Place back in the cooling chamber on ice.
  4. To prepare the reagents:
    1. Vortex at high speed 3 times, 1 sec each time (except for the enzyme).
    2. Pulse spin for 3 sec.
    3. Place in the cooling chamber.
  5. Preheat the Thermal Cycler lid, however leave the block at room temperature.

    Prepare the Nsp Ligation Master Mix (stage 5, steps 6-12)
  6. Keeping all reagents and tubes on ice, prepare the Nsp Ligation Master Mix by adding to the 2.0 ml Eppendorf tube the following reagents based on the volumes in Table 5.
    1. Adaptor Nsp
    2. T4 DNA Ligase Buffer (10x)
  7. Remove the T4 DNA Ligase from the freezer and immediately place in the cooler on ice.
  8. Pulse spin the T4 DNA Ligase for 3 sec.
  9. Immediately add the T4 DNA Ligase to the master mix; then place back in the cooler.
  10.  Vortex the master mix at high speed 3 times, 1 sec each time.
  11. Pulse spin for 3 sec.
  12.  Place the master mix on ice.

    Table 5. NspI Ligation Master Mix
    Reagent
    1 Sample
    48 Samples (15% Extra)
    T4 DNA Ligase Buffer (10x)
    2.5 μl
    150 μl
    Adaptor NspI (50 μM)
    0.75 μl
    45 μl
    T4 DNA Ligase (400 U/μl)
    2 μl
    120 μl
    Total
    5.25 μl
    315 μl

    Add Nsp Ligation Master Mix to Reactions (stage 5, steps 13-25)
  13. To add Nsp Ligation Master Mix to samples, use a single channel P100 pipet and aliquot 25 μl of Nsp Ligation Master Mix to each tube of the strip tubes on ice.
  14. Using a 12-channel P20 pipet, aliquot 5.25 μl of Nsp Ligation Master Mix to each reaction on the Nsp Digestion Stage Plate. The total volume in each well is now 25 μl.
    Reagent
    Volume
    Nsp Digested DNA
    19.75 μl
    Nsp Ligation Master Mix
    5.25 μl
    Total
    25 μl
  15. Seal the plate tightly with adhesive film.
  16. Vortex the center of the plate at high speed for 3 sec.
  17. Spin down the plate at 560 x g for 30 sec.
  18. Ensure that the thermal cycler lid is preheated.
  19. Load the plate onto the thermal cycler and run the GW5.0/6.0 Ligate program.
    GW5.0/6.0 Ligate Program
    Temperature
    Time
    16 °C
    180 min
    70 °C
    20 min
    4 °C
    Hold
  20. When the GW5.0/6.0 Ligate program is finished, remove the plate and spin it down at 560 x g for 30 sec.
  21. Place the plate in a cooling chamber on ice.
  22. Dilute each reaction as follows:
    1. Pour 10 ml AccuGENE water into the solution basin.
    2. Using a 12-channel P200 pipet, add 75 μl of the water to each reaction.
      The total volume in each well is 100 μl.
      Reagent
      Volume
      Nsp Ligated DNA
      25 μl
      AccuGENE water
      75 μl
      Total
      100 μl
  23. Seal the plate tightly with adhesive film.
  24. Vortex the center of the plate at high speed for 3 sec.
  25. Spin down the plate at 560 x g for 30 sec.
    Note: It is crucial to dilute the ligated DNA with AccuGENE water prior to PCR.

Stage 6: Nsp PCR
During this stage, you will transfer equal amounts of each Nsp ligated sample into four fresh 96-well plates; prepare the Nsp PCR Master Mix, and add it to each sample; place each plate on a thermal cycler and run the GW6.0 PCR program; and confirm the PCR by running 3 μl of each PCR product on a 2% TBE gel or an E-Gel® 96 2% agarose gel.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. TITANIUM Taq PCR Buffer
    2. dNTPs
    3. PCR Primer 002
    4. AccuGENE water
    5. Nsp ligated samples (if frozen)
    6. GC-Melt
    7. Solution basin
  2. Label the following tubes, then place in the cooling chamber or on ice, as appropriate:
    1. Four 96-well reaction plates labeled “P1”, “P2”, “P3”, “P4”
    2. One 50 ml Falcon tube labeled “PCR MM”
  3. Place enough cooling chambers for 5 plates and one cooler on ice.
    Note: Leave the TITANIUM Taq DNA Polymerase at –20 °C until ready to use.
  4. Prepare the Nsp ligated samples as follows:
    1. Vortex the center of the plate at high speed for 3 sec.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Label the plate Lig.
    4. Place back in the cooling chamber on ice.
  5. To prepare the reagents:
    1. Vortex at high speed 3 times, 1 sec each time (except for the enzyme).
    2. Pulse spin for 3 sec.
    3. Place in a cooling chamber.
  6. Preheat the Thermal Cycler lid, however leave the block at room temperature.

    Aliquot Nsp Ligated DNA to the PCR Plates (stage 6, steps 7-8)
  7. To aliquot Nsp ligated DNA to the PCR plates, working one row at a time and using a 12-channel P20 pipet, transfer 10 μl of each Nsp ligated sample to the corresponding well of each PCR plate (P1, P2, P3 and P4).
  8. Seal each plate with adhesive film, and leave in cooling chambers on ice.

    Prepare the Nsp PCR Master Mix (stage 6, steps 9-14)
  9. To prepare the Nsp PCR Master Mix, keep the 50 ml Falcon tube in the cooling chamber and add the reagents as shown in Table 6 (except for the Taq DNA polymerase).
  10. Remove the TITANIUM Taq DNA Polymerase from the freezer and immediately place in a cooler.
  11. Pulse spin the Taq DNA polymerase for 3 sec.
  12. Immediately add the Taq DNA polymerase to the master mix; then return the tube to the cooler on ice.
  13. Vortex the master mix at high speed 3 times, 1 sec each time.
  14. Pour the mix into the solution basin, keeping the basin on ice.

    Table 6. Prepare the Nsp PCR Master Mix
    Reagent
    For 1 Reaction
    4 PCR Plates (15% extra)
    AccuGENE water
    39.5 μl
    8.722 ml
    TITANIUM Taq PCR Buffer (10x)
    10 μl
    2.208 ml
    GC-Melt (5M)
    20 μl
    4.416 ml
    dNTP (2.5 mM each)
    14 μl
    3.091 ml
    PCR Primer 002 (100 μM)
    4.5 μl
    0.994 ml
    TITANIUM Taq DNA Polymerase (50x) (do not add until ready to aliquot master mix to ligated samples)
    2 μl
    0.442 ml
    Total
    90 μl
    19.873 ml

    Add Nsp PCR Master Mix to Samples (stage 6, steps 15-19)
  15. To add Nsp PCR Master Mix to samples, use a 12-channel P200 pipet and add 90 μl Nsp PCR Master Mix to each sample. To avoid contamination, change pipet tips after each dispense. The total volume in each well is 100 μl.
  16. Seal each reaction plate tightly with adhesive film.
  17. Vortex the center of each reaction plate at high speed for 3 sec.
  18. Spin down the plates at 560 x g for 30 sec.
  19. Keep the reaction plates in cooling chambers on ice until loaded onto the thermal cyclers.

    Load Nsp PCR Plates onto Thermal Cyclers (stage 6, steps 20-22)
  20. To load the plates and run the GW5.0/6.0 PCR program, ensure that the thermal cycler lids are preheated. The block should be at room temperature.
  21. Load each reaction plate onto a thermal cycler.
  22. Run the GW5.0/6.0 PCR program. The program varies depending upon the thermal cyclers you are using. The example used here is for GeneAmp® PCR System 9700, however another protocol can be found in the manufacturers’ instructions.
    GW5.0/6.0 PCR Program for GeneAmp® PCR System 9700
    Temperature
    Time
    Cycles
    94 °C
    3 min
    1x
    94 °C
    30 sec
    |
    60 °C
    45 sec
    30x
    68 °C
    15 sec
    |
    68 °C
    7 min
    1x
    4 °C
    Hold

    Volume: 100 μl
    Specify Maximum mode.

    Running Gels (stage 6, steps 23-33)
    The instructions below are for running 2% TBE gels. To ensure consistent results, take 3 μl aliquot from each PCR.
  1. When the GW5.0/6.0 PCR program is finished, remove each plate from the thermal cycler.
  2. Spin down plates at 560 x g for 30 sec.
  3. Place plates in cooling chambers on ice or keep at 4 °C.
  4. Label four fresh 96-well reaction plates P1Gel, P2Gel, P3Gel, and P4Gel.
  5. Aliquot 3 μl of 2x Gel Loading Dye to each well in rows A through D of the fresh, labeled PXGel plates.
  6. Using a 12-channel P20 pipet, transfer 3 μl of each PCR product from the 4 Nsp PCR plates to the corresponding plate, row and wells of the PXGel plates. Example: 3 μl of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel.
  7. Seal the PXGel plates.
  8. Vortex the center of each PXGel plate, then spin them down at 560 x g for 30 sec.
  9. Load the total volume from each well of each PXGel plate onto 2% TBE gels.
  10. Run the gels at 120 V for 40 min to 1 h.
  11. Verify that the PCR product distribution is between ~200 bp to 1,100 bp (example of gel below, Figure 2).


    Figure 2. Example of PCR products run on 2% TBE agarose gel at 120 V for 1 h. Average product distribution is between ~200 to 1,100 bp.

Stage 7: PCR Product Purification Using a Millipore Filter Plate
During this stage, you will pool the Sty and Nsp PCR reactions and purify DNA and transfer the purified products to a new 96-well plate.

  1. Prepare 75% EtOH by diluting ACS-grade or equivalent ethanol to 75% using AccuGENE water.
  2. Allow the Buffer EB to warm to room temperature prior to use.
  3. To prepare the manifold, connect the manifold and regulator to a suitable vacuum source able to maintain 20 to 24 in Hg. Leave the vacuum turned off at this time.
  4. Inspect the manifold for salt and other contaminants and clean if necessary.
  5. Place the vacuum flask trap below the level of the manifold.
  6. Place the standard collar on the manifold.

    Pool the PCR Products (stage 7, steps 7-14)
  7. If PCR products are frozen, thaw to room temperature on the bench top in plate holders, and if on thermal cyclers, remove them now.
  8. Vortex the center of each plate at high speed for 3 sec.
  9. Spin down each plate at 560 x g for 30 sec.
  10. Place each PCR plate in a plate holder on the bench top.
  11. Place a deep well pooling plate on the bench top.
  12. On each PCR plate, cut the seal between each row so that it can be removed one row at a time.
  13. Using a 12-channel P200 pipet set to 110 μl:
    1. Remove the seal to expose row A only on each PCR plate.
    2. Transfer the reactions from row A of each PCR plate to the corresponding wells of row A on the pooling plate.
      Sty PCR plates (3): 100 μl from each well = 300 μl/well
      Nsp PCR Plate (4): 100 μl from each well = 400 μl/well
      Total Volume Each Well of Pooling Plate = 700 μl/well
    3. Change your pipet tips.
    4. Change pipet tips after the PCR product from the same row of each PCR plate has been pooled on the pooling plate.
    5. Remove the seal from each PCR plate to expose the next row.
    6. Transfer each reaction from the same row of each PCR plate to the corresponding row and wells of the pooling plate.
    7. Repeat steps C., D. and E. until all of the reactions from each PCR plate are pooled.
  14. When finished, examine the wells of each PCR plate to ensure that all of the product has been transferred and pooled.
    Note: Be very careful when pooling PCR products. Avoid cross-contaminating neighboring wells with small droplets.

    Purify the Pooled PCR products (stage 7, steps 15-18)
  15. Mix the magnetic bead stock very well by vigorously shaking the bottle. Beads will settle overnight. Examine the bottom of the bottle and ensure that the solution appears homogenous.
  16. Pour or pipet 50 ml of magnetic beads to a solution basin. 1 ml of magnetic beads is required for each reaction. You can add in multiple batches if the solution basin is not large enough.
  17. Using a manual (not electronic) 12-channel P1200 pipet:
    1. Slowly add 1.0 ml of magnetic beads to each well of pooled PCR product.
    2. Mix well by pipetting up and down 5 times using the following technique:
    Mixing Technique:
    1. Depress the plunger and place the pipet tips into the top of the solution.
    2. Move the pipet tips down-aspirating at the same time-until the tips are near the bottom of each well.
    3. Raise the tips out of the solution.
    4. Place the pipet tips against the wall of each well just above each reaction, and carefully dispense the solution.
      Note: The solution is viscous and sticky. Pipet carefully to ensure that you aspirate and dispense 1 ml. Do not use an electronic pipet. Thorough mixing is critical to ensure that the PCR products bind to the beads.
    5. Change pipet tips for each row.
  18. Cover the plate to protect the samples from environmental contaminants and allow to incubate at room temperature for 10 min. You can use the lid from a pipet tip box to cover the wells.

    Transfer Reactions to a Filter Plate (stage 7, steps 19-21)
  1. Place the filter plate on the standard collar on the vacuum manifold.
  2. Using a 12-channel P1200 pipet, transfer each reaction from the pooling plate to the corresponding row and well of the filter plate.
    Note: You will need to pipet twice to transfer all of the solution from each well to the filter plate. The solution is viscous and sticky, so check to ensure that all of it has been transferred.
  3. Tightly seal the unused wells with a MicroAmp Clear Adhesive Film. To ensure a tight seal, cover 1/2 to 1/3 of the wells in row D as well. Unused wells must be sealed to ensure proper vacuum pressure.

    Purify the Reactions (stage 7, steps 22-30)
  4. To purify the reactions, turn on the vacuum to 20 to 24 in Hg and check the seals. Do not exceed 24 in Hg. Adjust the leak valve if necessary.
  5. Ensure that the unused wells are completely sealed, and cover the plate to protect it from environmental contaminants.
  6. Run the vacuum until all of the liquid has been pulled through the filter (approximately 40 to 50 min), then turn off the vacuum.
  7. Examine each well. There should be no standing liquid in any well, and the wells should appear dull (matte). Wet wells will look shiny. If any of the wells are still wet, put the plate back on the vacuum and continue filtering for up to 10 min (total ≤ 60 min).
  8. Using a 12-channel P1200 set to 900 μl:
    1. Add 900 μl of 75% EtOH to each reaction.
    2. Turn the vacuum on to 20 to 24 in Hg.
    3. Run the vacuum for approximately 1-2 min (or until the volume in the wells begins to decrease).
    4. Add another 900 μl of 75% EtOH to each reaction (for a total of 1.8 ml EtOH).
    5. Cover the plate.
    6. Run the vacuum until all of the liquid has been pulled through the filter (approximately 10 to 15 min), then turn off the vacuum.
  9. Examine each well. Again, there should be no standing liquid in any well, and the wells should appear dull (matte). Wet wells will look shiny. If any of the wells are still wet, put the plate back on the vacuum and continue filtering for up to 5 min (total ≤ 20 min).
  10. Remove any excess EtOH as follows:
    1. Blot the bottom of the plate on Kimwipes.
    2. Wipe the bottom of the plate with a clean Kimwipe.
  11. Return the filter plate to the manifold and turn on the vacuum for an additional 10 min only. Do not exceed 10 min. Less than 10 min is ok if no excess ethanol is present in the wells or on the underside of the filter plate.
    Note: The purpose of this step is to remove excess EtOH so that it is not carried over into the eluate. Ten minutes is sufficient for this purpose. Leaving the vacuum on for more than 10 min may over-dry the beads which may inhibit elution of the purified DNA.
  12. Turn off the vacuum, and blot the bottom of the plate on Kimwipes to remove any remaining EtOH.

    Elute the Purified Reactions (stage 7, steps 31-44)
  13. To elute the purified reactions, attach the elution catch plate to the bottom of the filter plate using 2 strips of lab tape. The filter and elution plate assembly is now referred to as the plate stack.
    Note: Do not completely seal with tape. Product will not elute if sealed.
  14. Pour or pipet 3 ml of Buffer EB to a solution basin.
  15. Using a 12-channel P200 pipet, add 55 μl of Buffer EB to each well. For accurate pipetting, pre-wet pipet tips with EB before dispensing. Dispense as close to the beads as possible without touching them. Buffer EB should be applied directly on top of the beads.
  16. Tap the plate stack to move all Buffer EB onto the filter at the bottom of the wells.
  17. Using an adhesive film, tightly seal the filter plate.
  18. Place the plate stack on a Jitterbug for 10 min at setting 5.
  19. Inspect each well to verify that the beads are thoroughly resuspended.
  20. The beads must be thoroughly resuspended in Buffer EB so that the DNA can come off the beads.
  21. Remove the plate stack from the Jitterbug and remove the adhesive seal.
  22. Continue elution on the vacuum manifold as follows:
    1. Remove the standard collar from the manifold.
    2. Place the plate stack inside the manifold.
    3. Place the standard collar around the plate stack (Figure 4.13 on page 97).
    4. Seal the empty wells with adhesive film.
    5. Turn the vacuum on to 20 to 24 in Hg and ensure that a seal has been formed between the collar and the base of the manifold.
    6. Ensure that the unused wells are completely sealed.
    7. Cover the plate stack to protect it from environmental contaminants.
    8. Run the vacuum until all of the liquid has been pulled through the filter (approximately 5 to 15 min).
    9. Turn off the vacuum.
  23. Examine each well. Again, there should be no standing liquid in any well, and the wells should appear dull (matte). Wet wells will look shiny. If any of the wells are still wet, continue filtering for up to 15 additional min.
  24. Seal the plate stack with an adhesive film, and spin it down at room temperature for 5 min at 1,400 x g.
  25. Remove the elution catch plate from the filter plate.
  26. Using a 12-channel P200 pipet, transfer 45 μl of eluate to a new PCR plate for fragmentation.

Stage 8: Quantitation
During this stage, you will prepare one dilution of each PCR product in optical plates. You will then quantitate the diluted PCR products.
Note: This protocol has been optimized using a UV spectrophotometer plate reader for quantitation. The NanoDrop® will give different quantitation results. This protocol has not been optimized for use with this instrument. In addition, the NanoDrop quantifies a single sample at a time and is not amenable to 96-well plate processing.

  1. Turn on the spectrophotometer now and allow it to warm for 10 min before use.
  2. Place the following on the bench top
    1. Optical plate
    2. Solution basin
    3. AccuGENE water
  3. Label the optical plate “OP”.
  4. Prepare the purified, eluted PCR product plate as follows:
    1. If the plate was frozen, allow it to thaw in a cooling chamber on ice.
    2. Spin down the plate at 560 x g for 30 sec.
    3. Place the plate on the bench top.
  5. To prepare diluted aliquots of the purified samples, pour 15 ml of room temperature AccuGENE water into the solution basin.
    Note: One row of wells on the optical plate are used as blanks and contain AccuGENE water only.
  6. Using a 12-channel P200 pipet aliquot 198 μl of water to each well in rows A through E of the optical plate.
  7. Using a 12-channel P20 pipet:
    1. Transfer 2 μl of each purified PCR product from rows A through D of the purified sample plate to the corresponding rows and wells of the optical plate. Row E remains water only and will serve as a blank.
    2. Pipet up and down 2 times after each transfer to ensure that all of the product is dispensed.
    3. Examine the pipet tips and aliquots before and after each dispense to ensure that exactly 2 μl has been transferred. The result is a 100-fold dilution.
  8. Set a 12-channel P200 pipet to 180 μl.
  9. Mix each sample by pipetting up and down 3 times. Be careful not to scratch the bottom of the plate, or to introduce air bubbles.
  10. To quantitate the diluted PCR product, measure the OD of each PCR product at 260, 280 and 320 nm. OD280 and OD320 are used as process controls.
  11. Determine the OD260 measurement for the water blank and average.
  12. Determine the concentration of each PCR product as follows:
    1. Take 1 OD reading for every sample.
      OD = (sample OD)-(average water blank OD)
    2. Calculate the undiluted sample concentration for each sample using the Sample OD:
      Sample concentration in μg/μl = OD x 0.05 μg/μl x 100
      Apply the convention that 1 absorbance unit at 260 nm equals 50 μg/ml (equivalent to 0.05 μg/μl) for double-stranded PCR products. This convention assumes a path length of 1 cm. Consult your spectrophotometer handbook for further information.

Stage 9: Fragmentation
During this stage, the purified PCR products are fragmented using Fragmentation Reagent. You will first dilute the Fragmentation Reagent by adding the appropriate amount of Fragmentation Buffer and molecular biology-grade water. You will then quickly add the diluted reagent to each reaction, place the plate onto a thermal cycler, and run the GW6.0 Fragment program. Once the program is finished, you will verify fragmentation by running 1.5 μl of each reaction on a 4% TBE gel or an E-Gel 48 4% agarose gel.

  1. Thaw/place the following reagents on ice (approximately 20 min):
    1. Fragmentation Buffer
    2. AccuGENE water
  2. Prepare the Fragmentation Buffer as follows:
    1. Vortex 3 times, 1 sec each time.
    2. Pulse spin for 3 sec.
    3. Place the buffer in the cooling chamber on ice.
  3. Label the following tubes, then place in the cooling chamber or on ice, as appropriate:
    1. Two strips of 12 tubes each: one labeled ‘Buffer’ and one labeled ‘FR’
    2. One 2.0 ml Eppendorf tube labeled ‘Frag MM’
    3. Plate of purified PCR product from the previous stage
  4. Power on the thermal cycler and preheat the block to 37 °C. Allow it to heat for 10 min before loading samples.
  5. To prepare the samples for Fragmentation, aliquot 28 μl of 10x Fragmentation Buffer to each tube of the strip tubes labeled Buffer.
  6. Using a 12-channel P20 pipet, add 5 μl of Fragmentation Buffer to each sample in the 96-well reaction plate. Check your pipet tips each time to ensure that all of the buffer has been dispensed. The total volume in each well is now 50 μl.
  7. Read the Fragmentation Reagent tube label and record the concentration.
  8. Dilute the Fragmentation Reagent to 0.1 U/μl as described below using the appropriate recipe from Table 7.

    Table 7. Dilution Recipes for the Fragmentation Reagent
    Reagent
    Fragmentation Reagent Concentration
    2 U/μl
    2.25 U/μl
    2.5 U/μl
    2.75 U/μl
    3 U/μl
    AccuGENE water
    306 μl
    308 μl
    309.6 μl
    310.9 μl
    312 μl
    10x Fragmentation Buffer
    36 μl
    36 μl
    36 μl
    36 μl
    36 μl
    Fragmentation Reagent
    18 μl
    16 μl
    14.4 μl
    13.1 μl
    12 μl
    Total
    (enough for 48 samples)
    360 μl
    360 μl
    360 μl
    360 μl
    360 μl

  9. To the 2.0 ml Eppendorf tube on ice, add the AccuGENE water and Fragmentation Buffer. Allow to cool on ice for 5 min.
  10. Remove the Fragmentation Reagent from the freezer and immediately pulse spin for 3 sec then place in a cooler.
  11. Add the Fragmentation Reagent to the 1.5 ml Eppendorf tube.
  12. Vortex the diluted Fragmentation Reagent at high speed 3 times, 1 sec each time.
  13. Pulse spin for 3 sec and immediately place on ice.
  14. To add diluted Fragmentation Reagent to the samples, quickly and on ice, aliquot 28 μl of diluted Fragmentation Reagent to each tube of the strip tubes labeled FR. Avoid introducing air bubbles at the bottom of the strip tubes to ensure the accurate transfer of 5 μl diluted DNA to each sample.
  15. Using a 12-channel P20 pipet, add 5 μl of diluted Fragmentation Reagent to each sample. Do not pipet up and down.
    Sample with Fragmentation Buffer
    50 μl
    Diluted Fragmentation Reagent (0.1 U/μl)
    5 μl
    Total
    55 μl
  16. Seal the plate and inspect the edges to ensure that it is tightly sealed.
  17. Vortex the center of the plate at high speed for 3 sec.
  18. Place the plate in a chilled plastic plate holder and spin it down at 4 °C at 560 x g for 30 sec.
  19. Immediately load the plate onto the pre-heated block of the thermal cycler (37 °C) and run the GW5.0/6.0 Fragment program.
    GW5.0/6.0 Fragment Program
    Temperature
    Time
    37 °C
    35 min
    95 °C
    15 min
    4 °C
    Hold
  20. Discard any remaining diluted Fragmentation Reagent. Diluted Fragmentation Reagent should never be reused.
  21. Remove the plate from the thermal cycler.
  22. Spin down the plate at 560 x g for 30 sec, and place in a cooling chamber on ice.
  23. Dilute 1.5 μl of each fragmented PCR product with 4 μl gel loading dye.
  24. Run on 4% TBE gel with the BioNexus All Purpose Hi-Lo ladder at 120 V for 30 min to 1 h.
  25. Inspect the gel (example of gel below, Figure 3).


    Figure 3. Typical example of fragmented PCR products run on 4% TBE agarose gel at 120 V for 30 min to 1 h. Average fragment size is < 180 bp.

Stage 10: Labeling
During this stage, 48 fragmented samples are labeled using the DNA Labeling Reagent.

  1. Prepare Your Work Area
    To prepare the work area:
    1. Place a double cooling chamber and a cooler on ice.
    2. Prepare the reagents as follows:
      1. Vortex each reagent at high speed 3 times, 1 sec each time.
      2. Pulse spin for 3 sec; then place in the cooling chamber.
        Reagents required for Stage 10: Labeling
        Quantity
        Reagent
        1 vial
        DNA Labeling Reagent (30 mM)
        1 vial Terminal
        Deoxynucleotidyl Transferase (TdT; 30 U/μl)
        1 vial Terminal
        Deoxynucleotidyl Transferase Buffer (TdT Buffer; 5x)
  2. Thaw/place the following reagents on ice (approximately 20 min):
    1. 5x TdT Buffer
    2. DNA Labeling Reagent
  3. Label one 15 ml centrifuge tube MM, and place on ice.
  4. Label and place the following in the cooling chamber:
    1. One strip of 12 tubes labeled MM
    2. Plate of fragmented reactions from the previous stage
  5. Power on the thermal cycler and preheat the block to 37 °C. Allow it to heat for 10 min before loading samples.
  6. To prepare the Labeling Master Mix, add the following to the 15 ml centrifuge tube on ice using the volumes shown in Table 8.
    1. 5x TdT Buffer
    2. DNA Labeling Reagent

    Table 8. Labeling Master Mix
    Reagent
    1 Sample
    48 Samples (15% extra)
    TdT Buffer (5x)
    14 μl
    772.8 μl
    DNA Labeling Reagent (30 mM)
    2 μl
    110.4 μl
    TdT enzyme (30 U/μl)
    3.5 μl
    193.2 μl
    Total
    19.5 μl
    1076.4 μl

  7. Remove the TdT enzyme from the freezer and immediately place in the cooler.
  8. Pulse spin the enzyme for 3 sec; then immediately place back in the cooler.
  9. Add the TdT enzyme to the master mix.
  10. Vortex the master mix at high speed 3 times, 1 sec each time.
  11. Pulse spin for 3 sec.
  12. To add the Labeling Master Mix to the samples, and while keeping samples in the cooling chamber and all tubes on ice when making additions, aliquot 89 μl of Labeling Master Mix to each tube of the strip tubes.
  13. Add the Labeling Master Mix as follows:
    1. Using a 12-channel P20 pipet, aliquot 19.5 μl of Labeling Master Mix to each sample.
    2. Pipet up and down one time to ensure that all of the mix is added to the samples. The total volume in each well is now 73 μl.
    Reagent
    Volume
    Fragmented DNA (less 1.5 μl for gel analysis)
    53.5 μl
    Labeling Mix
    19.5 μl
    Total
    73 μl
  14. Seal the plate tightly with adhesive film.
  15. Vortex the center of the plate at high speed for 3 sec.
  16. Spin down the plate at 560 x g for 30 sec.
  17. Place the plate on the pre-heated thermal cycler block, and run the GW5.0/6.0 Label program.
    GW5.0/6.0 Label Program
    Temperature
    Time
    37 °C
    4 h
    95 °C
    15 min
    4 °C
    Hold
  18. When the GW5.0/6.0 Label program is finished:
    1. Remove the plate from the thermal cycler.
    2. Spin down the plate at 560 x g for 30 sec.

Stage 11: Target Hybridization
During this stage, each reaction is loaded onto a Genome-Wide Human SNP Array 6.0.

  1. To prepare 1,000 ml of 12x MES Stock Solution: (1.25 M MES, 0.89 M [Na+]), combine:
    1. 70.4 g MES hydrate
    2. 193.3 g MES sodium salt
    3. 800 ml AccuGENE® water
  2. Mix and adjust volume to 950 ml.
  3. Test the pH, which should be between 6.5 and 6.7.
  4. Adjust the pH so it falls between 6.5 and 6.7.
  5. Adjust the volume to 1,000 ml.
  6. Filter the solution through a 0.2 μm filter.
  7. Protect from light using aluminum foil and store at 4 °C.
  8. Preheat the hybridization ovens by turning each oven on and setting the temperature to 50 °C.
  9. Set the rpm to 60.
  10. Turn the rotation on and allow to preheat for 1 h before loading arrays.
  11. If the labeled samples from the previous stage were frozen, thaw the plate on the bench top.
  12. Vortex the center of the plate at high speed for 3 sec.
  13. Spin down the plate at 560 x g for 30 sec.
  14. Place in a cooling chamber on ice.
  15. Labeled samples must be placed in a Bio-Rad unskirted 96-well plate.
  16. Power on the thermal cycler to preheat the lid. Leave the block at room temperature.
  17. To prepare the arrays, unwrap the arrays and place on the bench top, septa-side up.
  18. Mark each array with a meaningful designation (e.g., a number) to ensure that you know which sample is loaded onto each array.
  19. Allow the arrays to warm to room temperature by leaving on the bench top 10 to 15 min.
  20. Insert a 200 μl pipet tip into the upper right septum of each array.
  21. To prepare the Hybridization Master Mix to the 50 ml centrifuge tube, add the reagents in the order shown in Table 9 and mix well.
    Note: DMSO addition: pipet directly into the solution of other reagents. Avoid pipetting along the side of the tube.

    Table 9. Hybridization Master Mix
    Reagent
    1 Array
    48 Arrays (15% extra)
    MES (12x; 1.25 M)
    12 μl
    660 μl
    Denhardt’s Solution (50x)
    13 μl
    715 μl
    EDTA (0.5 M)
    3 μl
    165 μl
    HSDNA (10 mg/ml)
    3 μl
    165 μl
    OCR, 0100
    2 μl
    110 μl
    Human Cot-1 DNA® (1 mg/ml)
    3 μl
    165 μl
    Tween-20 (3%)
    1 μl
    55 μl
    DMSO (100%)
    13 μl
    715 μl
    TMACL (5 M)
    140 μl
    7.7 ml
    Total
    190 μl
    10.45 ml

    Using a GeneAmp® PCR System 9700
  22. To add Hybridization Master Mix and denature the samples, pour 11 ml Hybridization Master Mix into a solution basin.
  23. Using a 12-channel P200 pipet, add 190 μl of Hybridization Master Mix to each sample on the Label Plate. Total volume in each well is 263 μl.
  24. Seal the plate tightly with adhesive film.
  25. Vortex the center of the plate for 30 sec.
  26. Spin down the plate at 560 x g for 30 sec.
  27. Cut the adhesive film between each row of samples. Do not remove the film.
  28. Place the plate onto the thermal cycler and close the lid.
  29. Run the GW5.0/6.0 Hyb program.
    GW5.0/6.0 Hyb Program
    Temperature
    Time
    95 °C
    10 min
    49 °C
    Hold
    Note: The following procedure requires 2 operators working simultaneously. Operator 1 loads the samples onto the arrays; Operator 2 covers the septa with Tough-Spots and loads the arrays into the hybridization ovens.
    30a.Operator 1 Tasks (after completing stage 11, step 29)

    1. When the plate reaches 49 °C, slide back the lid on the thermal cycler enough to expose the first row of samples only.
    2. Remove the film from the first row.
    3. Using a single-channel P200 pipet, remove 200 μl of denatured sample from the first well.
    4. Immediately inject the sample into an array.
    5. Pass the array to Operator 2.
    6. Remove 200 μl of sample from the next well and immediately inject it into an array.
    7. Pass the array to Operator 2.
    8. Repeat this process one sample at a time until the entire row is loaded.
    9. Place a fresh strip of adhesive film over the completed row.
    10. Slide the thermal cycler lid back to expose the next row of samples.
    11. Repeat steps 3 through 10 until all of the samples have been loaded onto arrays.
    30b. Operator 2 Tasks (after completing stage 11, step 29 )
    1. Cover the septa on each array with a Tough-Spot.
    2. For every 4 arrays:
      1. Load the arrays into an oven tray evenly spaced.
      2. Immediately place the tray into the hybridization oven.
    Note: Do not allow loaded arrays to sit at room temperature for more than approximately 1.5 min. Ensure that the oven is balanced as the trays are loaded, and ensure that the trays are rotating at 60 rpm at all times. Because you are loading 4 arrays per tray, each hybridization oven will have a total of 32 arrays.

    Notes for Operators 1 and 2:
    1. Load no more than 32 arrays in one hybridization oven at a time.
    2. All 48 samples should be loaded within 1 h.
    3. Store the remaining samples and any samples not yet hybridized in a tightly sealed plate at -20 °C.
    4. Allow the arrays to rotate at 50 °C, 60 rpm for 16 to 18 h.
    5. Allow the arrays to rotate in the hybridization ovens for 16 to 18 h at 50 °C and 60 rpm. This temperature is optimized for this product, and should be stringently followed.
    6. Continue on to washing, staining and scanning arrays.

Recipes

  1. Clontech TITANIUMTM DNA Amplification Kit
    1. dNTPs (2.5 mM each)
    2. GC-Melt (5 M)
    3. TITANIUMTM Taq DNA Polymerase (50x)
    4. TITANIUMTM Taq PCR Buffer (10x)
  2. 12x MES Stock Solution [1.25 M MES, 0.89 M (Na+)]
    1. 70.4 g MES hydrate
    2. 193.3 g MES sodium salt
    3. 800 ml AccuGENE® water

Acknowledgments

Funding: Swiss National Foundation 144082 and Gertrude von Meissner Foundation grant.

References

  1. Komura, D., Shen, F., Ishikawa, S., Fitch, K. R., Chen, W., Zhang, J., Liu, G., Ihara, S., Nakamura, H., Hurles, M. E., Lee, C., Scherer, S. W., Jones, K. W., Shapero, M. H., Huang, J. and Aburatani, H. (2006). Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays. Genome Res 16(12): 1575-1584.

简介

为了评估基因组变异,可以鉴定个体携带的单核苷酸多态性(SNP)。 使用Affymetrix全基因组人类SNP测定,可以在单个阵列上评估906,600个SNP。 这个协议是GeneChip Mapping 500K数组集的下一个迭代,它直接基于制造商的协议,并显示了与这里非常相似的步骤:http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf

关键字:单核苷酸多态性, 阵列, 人体细胞

材料和试剂

  1. 96孔V-底板,洗脱捕获物(Greiner Bio-One GmbH,目录号:651101)
  2. Greiner UV星透明,96孔(Greiner Bio-One GmbH,目录号:82050-788) 注意:目前,它是"VWR国际,目录号:82050-788"。
  3. 多孔板96孔非敲击PCR板(Bio-Rad Laboratories,目录号:MLP-9601)
  4. 多屏深孔板(Merck Millipore Corporation,目录号:MDRLN0410)
  5. 2.4ml深孔存储板/池板(Greiner Bio-One GmbH,目录号:780280)
  6. 100ml溶液池(VWR International,Labcor TM ,目录号:730-014)
  7. 55 V溶液盆(VWR International,Labcor ,目录号:730-004)
  8. 15ml离心管(VWR International,目录号:20171-020)
  9. 50ml离心管(VWR International,目录号:21008-178)
  10. 2.0ml Eppendorf管(VWR International,目录号:20901-540)
  11. 0.2ml 12-条管(VWR International,目录号:21008-940)
  12. Lab磁带
  13. 坚韧的®
  14. Microseal'B'粘合密封(Bio-Rad Laboratories,型号:MSB1001)
  15. 施加的MicroAmp透明粘合剂膜(Biosystems,目录号:4306311)
    注意:目前,它是"Thermo Fisher Scientific,Applied Biosystems TM,目录号:4306311"。
  16. Kimwipes(根据需要来源)
  17. 移液器吸头适合移液器(根据需要来源)
  18. AccuGENE 水,分子生物学级(Affymetrix,目录号:71786)
  19. GenElute Mammalian Genomic DNA Miniprep(Sigma-Aldrich,目录号:G1N70)
  20. Affymetrix SNP 6 Core Reagent Kit,100反应(Affymetrix,目录号:901706)
    1. 10x Sty I缓冲液/NE缓冲液3(New England Biolabs)
    2. T4 DNA连接酶(400U /μl)(New England Biolabs)
    3. T4 DNA连接酶缓冲液(10x)(New England Biolabs)
    4. 适配器,sty(50μM)
    5. PCR引物002(100μM)
    6. 10x Nsp I缓冲液/NE缓冲液2(New England Biolabs)
    7. Nsp I(10U /μl)(New England Biolabs)
    8. 适配器,nsp(50μM)
    9. 洗脱缓冲液(缓冲液EB)
    10. 碎片缓冲区(10x)
    11. 碎裂试剂(DNase I)
    12. DNA标记试剂
    13. 末端脱氧核苷酸转移酶(TdT)(30U /μl)
    14. 末端脱氧核苷酸转移酶缓冲液(TdT缓冲液)(5x)
    15. 寡核苷酸对照试剂(OCR),0100
    16. 10 mg/ml BSA(100x)
    17. 杂交主混合物
  21. DNA标记(Bionexus,目录号:BN2050)
  22. 2%TBE凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:G8008-02)
  23. 凝胶加载溶液(Faster Better Media,目录号:SB5N-8)
  24. 乙醇(Sigma-Aldrich,目录号:459844)
  25. 50ml磁珠(AMPure,目录号:A63881)
    注意:在网站上命名为"Agencourt AMPure XP-PCR Purification"
  26. 4%TBE凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:G8008-04)
  27. MES水合物(Sigma-Aldrich,目录号:M8250)
  28. MES钠盐(Sigma-Aldrich,目录号:M3885) 注意:所有目录号都列在Affymetrix数据表中( http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf )。
  29. Clontech TITANIUM™DNA扩增试剂盒(Clontech,目录号:639240)(参见配方)
  30. 12x MES库存解决方案(参见配方)

设备

  1. 应用生物系统部门
    1. 2720 Thermal Cycler
    2. GeneAmp PCR System 9700
    3. GeneAmp PCR系统9700(银块或镀金银块)
  2. 48基因组范围人类SNP阵列6.0(每个样品一个阵列)
  3. GeneChip ® Hybridization Oven 640
  4. Jitterbug
  5. 分光光度计读数器
  6. 真空歧管(Merck Millipore Corporation)
  7. Vortexer
  8. 冷却器(冷却至-20°C)
  9. 冷却室(冷却至4℃)(不冻结)
  10. 冷却室(双重,冷却至4℃)(不冻结)
  11. 冰桶,装满冰
  12. 标记,细点,永久性
  13. 微量离心机
  14. 吸管,12通道P1200
  15. 吸管,12通道P200
  16. 吸管,12通道P20(精确到±5%以内)
  17. 吸管,血清学
  18. 吸管,单通道P1000
  19. 移液器,单通道P200
  20. 具有深井容量(54mmH×160g)的板式离心机
  21. 光学板
  22. 离心机板支架
    注意:
    1. 只使用此处列出的PCR板,粘性膜和热循环仪。
    2. 所有相关设备编号都列在Affymetrix数据表中。
    http://media.affymetrix.com/support/downloads/manuals /genomewidesnp6_manual.pdf

程序

基因组DNA平板制备

  1. 使用提取方案从感兴趣的细胞提取DNA,产生高质量,无污染物的没有抑制剂的DNA( e GenElute Mammalian Genomic DNA Miniprep)。
    注意:当使用分光光度计检查光密度时,DNA在260/280比率下应该在1.8左右,在260/230比例时应该在2.0左右。
  2. 通过高速涡旋3秒彻底混合基因组DNA。
  3. 确定每个样品的浓度(例如 Nanodrop)。
  4. 使用还原的EDTA TE缓冲液稀释每个样品至50ng /μl
  5. 通过高速涡旋混合稀释的DNA 3秒
  6. 在高速下涡旋基因组DNA平板10秒,然后在560×g下旋转30秒。
  7. 等分5微升的每个DNA到两个96孔反应板的相应的孔。 5μl的50ng /μl工作原液相当于每孔250ng基因组DNA。 该协议需要每个样品两个重复:一个用于Nsp,一个用于处理Sty
  8. 用胶膜密封每个板。

阶段1:病毒限制性酶消化
在此阶段,基因组DNA被Sty I限制酶消化。

  1. 解冻/将下列试剂放在冰上:
    1. NE缓冲区3
    2. BSA
    3. 基因组DNA(如果冻存)
    4. AccuGENE水
  2. 标记以下管,然后放入冷却室:
    1. 标有"Dig"的12条管的一条。
    2. 一个2.0ml Eppendorf管标记"Dig MM"。
  3. 用基因组DNA制备板,如下:
    1. 以高速旋转板的中心3秒。
    2. 沿560英尺x g 旋转板30秒。
    3. 放回冷却室在冰上。
  4. 准备试剂(除了酶)如下:
    1. 涡旋3次,每次1秒
    2. 脉冲旋转3秒。
    3. 放置在冷却室中。
  5. 预热热循环仪盖,但在室温下离开块。
    注意:将STY I酶保持在-20°C,直到准备使用。
  6. 通过将以下试剂的体积加入到如表1中的2.0ml Eppendorf管中来制备Sty消化主混合物:
    1. AccuGENE水
    2. NE缓冲区3
    3. BSA
  7. 从冷冻机中取出Sty I酶,立即放入冷却器中。
  8. 脉冲旋转酶3秒。
  9. 立即将酶添加到主混合物中,然后将剩余的酶放回冷却器中
  10. 高速旋转主混合物3次,每次1秒
  11. 脉冲旋转3秒。
  12. 置于冷却室中。
  13. 将任何剩余的酶返回到冰箱
  14. 立即进行添加Sty Digestion主混合物到样品
    表1. Sty I Digestion Master Mix
    试剂
    1样本
    48份样品(15%额外)
    AccuGENE ®
    11.55微升
    637.6微升
    NE缓冲区3(10x)
    2微升
    110.4μl
    BSA(100×; 10mg/ml) 0.2μl
    11微升
    Sty I(10 U /μl)
    1微升
    55.2μl
    总计
    14.75微升
    814.2微升

    将Sty Digestion Master Mix添加到样品中(第1阶段,第15-22步)
  15. 要将Sty Digestion Master Mix添加到样品中,请使用单通道P200移液器,将67μlSty Digestion Master Mix等分到标有 Dig 的条带管的每个管。
  16. 使用12通道P20移液管,在冰上冷却室中的每个DNA样品中加入14.75μl的Sty Digestion Master Mix。 每个孔中的总体积现在为19.75μl
    试剂

    基因组DNA(50 ng /μl)
    5微升
    消化主混合物
    14.75微升
    总量
    19.75微升
  17. 用胶膜密封板。
  18. 以高速旋转板的中心3秒。
  19. 沿560英尺x g 旋转板30秒。
  20. 确保热循环仪的盖子已预热。
  21. 将板装载到热循环仪上,运行GW5.0/6.0 Digest程序GW5.0/6.0文摘计划
    温度
    时间
    37°C
    120分钟
    65℃
    20分钟
    4°C
    暂停
  22. 程序完成后,取出版面并以560 x g 旋转30秒。

阶段2:结婚连结
在此阶段,使用Sty适配器连接消化的样品。

  1. 解冻/将以下试剂放在冰上(约20分钟):
    1. 适配器垫片I
    2. T4 DNA连接酶缓冲液(10x)
    3. Sty消化样品(如果冷冻)
    4. AccuGENE水
    注意:将T4 DNA连接酶置于-20°C,直到准备使用。
  2. 标记以下管,然后放入冷却室:
    1. 一条12条标有"Lig"的管子
    2. 标记为"Lig MM"的2.0ml Eppendorf管
    3. 解决方案盆地
  3. 按如下方法制备消解样品:
    1. 以高速旋转板的中心3秒。
    2. 沿560英尺x g 旋转板30秒。
    3. 放回冷却室在冰上。
  4. 准备试剂:
    1. 高速涡旋3次,每次1秒(除了酶)
    2. 脉冲旋转3秒。
    3. 放置在冷却室中。
  5. 预热热循环仪盖,但在室温下离开块。 在样品装入前,盖子必须预热。
    注意:T4 DNA连接酶缓冲液(10x)含有ATP,应在冰上解冻。 在使用前根据需要涡旋缓冲液,以确保沉淀物重新悬浮,并且缓冲液是澄清的。 避免每个供应商的说明有多个冻融循环。

    准备Sty连接主混合物(阶段2,步骤6-13)
  6. 将所有试剂和管保持在冰上,通过向2.0ml Eppendorf管中加入基于表2中所示体积的以下试剂来制备Sty Ligation Master Mix:
    1. 适配器垫片I
    2. T4 DNA连接酶缓冲液(10x)

    表2. Sty I Ligation Master Mix
    试剂
    1样本
    48份样品(多加15%)
    T4连接酶缓冲液(10x)
    2.5μl
    150μl
    适配器Sty I(50μM)
    0.75μl
    45微升
    T4 DNA连接酶(400U /μl) 2微升
    120微升
    总计
    5.25微升
    315微升

  7. 从冰箱中取出T4 DNA连接酶,立即置于冰箱中冷藏。
  8. 脉冲旋转T4 DNA连接酶3秒。
  9. 立即将T4 DNA连接酶加入到主混合物中; 然后放回冷却器。
  10. 高速旋转主混合物3次,每次1秒
  11. 脉冲旋转3秒。
  12. 将主混合物置于冰上。
  13. 立即进行添加Sty连接主混合物到反应。

    将Sty Ligation主混合添加到反应中(第2阶段,第14-27步)
  14. 使用单通道P100移液管将Sty Ligation Master Mix加入样品中,在冰上将25μlSty Ligation Master Mix等分到试管的每个管中。
  15. 使用12通道P20移液器,等分5.25μl的Sty连接主混合物在Sty消化阶段板上的每个反应。 每个孔中的总体积现在为25μl
    试剂

    Sty消化的DNA
    19.75微升
    Sty Ligation Master Mix
    5.25微升
    总计
    25μl
  16. 用胶膜密封板。
  17. 以高速旋转板的中心3秒。
  18. 在560秒向下旋转盘子 30秒。
  19. 确保热循环仪盖已预热。
  20. 将板载入热循环仪并运行GW5.0/6.0 Ligate程序。
    GW5.0/6.0升级程序
    温度
    时间
    16°C

    70℃
    20分钟
    4°C
    暂停
  21. 当GW5.0/6.0 Ligate程序完成后,取出板并在560 x g下旋转30秒。
  22. 将板放在冰上的冷却室中。
  23. 稀释每个反应如下:
    1. 将10 ml AccuGENE水倒入溶液池。
    2. 使用12通道P200移液器,为每个反应添加75μl的水。
    每个孔中的总体积为100μl
    试剂

    Sty连接的DNA
    25μl
    AccuGENE水
    75微升
    总计
    100微升
  24. 用胶膜密封板。
  25. 以高速旋转板的中心3秒。
  26. 沿560英尺x g 旋转板30秒。
  27. 将板在冰上的冷却室中储存高达60分钟。 如果不直接进行下一步,将板存储在-20°C 注意:在PCR之前,用AccuGENE水稀释连接的DNA是至关重要的

阶段3:Sty PCR
在此阶段,您将等量的每个Sty连接的样品转移到三个新鲜的96孔板中; 准备Sty PCR主混合物,并将其加入每个样品; 将每个板放在热循环仪上并运行GW6.0 PCR程序; 并通过在2%TBE凝胶或E-Gel 962%琼脂糖凝胶上运行3μl每种PCR产物来确认PCR。

  1. 解冻/将以下试剂放在冰上(约20分钟):
    1. TITANIUM Taq PCR Buffer
    2. dNTPs
    3. PCR引物002
    4. 结扎样品(如果冷冻)
    5. AccuGENE水
    6. GC-Melt
    7. 解决方案盆地
    注意:将TITANIUM Taq DNA聚合酶留在-20°C,直到准备使用。
  2. 标记以下管,然后根据需要放置在冷却室或冰上:
    1. 标记为"P1","P2","P3"的三个96孔反应板
    2. 一个标记为"PCR MM"的50ml Falcon管
  3. 按照以下步骤准备Sty连接的样品:
    1. 以高速旋转板的中心3秒。
    2. 沿560英尺x g 旋转板30秒。
    3. 标记板Lig。
    4. 放回冷却室在冰上。
  4. 准备试剂:
    1. 高速涡旋3次,每次1秒(除了酶)
    2. 脉冲旋转3秒。
    3. 放置在冷却室中。
  5. 预热热循环仪盖,但在室温下离开块。

    将PCR样品连接到PCR板(阶段3,步骤6-7)
  6. 每次使用12通道P20移液管一行,将10μl每个Sty连接的样品转移到每个PCR板的相应孔中。
  7. 用粘合剂膜密封每个板,并在冷却室中在冰上离开
    准备Sty PCR预混液(第3阶段,第8-13步)
  8. 为了制备Sty PCR主混合物,将50ml Falcon管保持在冷却室中,并加入如表3所示的试剂(除了Taq DNA聚合酶)。

    表3.48个样品的Sty PCR主混合物
    试剂
    1反应
    3 PCR板,
    48每个样板取样
    (15%额外)
    AccuGENE水
    39.5微升
    6.541 ml
    TITANIUM Taq PCR缓冲液(10x)
    10微升
    1.656 ml
    GC-Melt(5M)
    20微升
    3.312 ml
    dNTP(各2.5mM) 14μl
    2.318 ml
    PCR引物002(100μM) 4.5微升
    0.745 ml
    TITANIUM DNA聚合酶(50x)(在准备将等分试样混合到连接的样品前不要添加)
    2微升
    0.331毫升
    总计
    90微升
    14.903 ml

  9. 从冷冻机中取出TITANIUM Taq DNA聚合酶,立即置于冷却器中
  10. 脉冲旋转Taq DNA聚合酶3秒
  11. 立即将Taq DNA聚合酶加入到主混合物中; 然后在冰上将管子放回冷却器
  12. 高速旋转主混合物3次,每次1秒
  13. 将混合物倒入溶液盆中,将盆保持在冰上。

    将Sty PCR Master Mix添加到样品中(第3阶段,第14-18步)
  14. 要向样品中加入Sty PCR Master Mix,使用12通道P200移液管,并向每个样品加入90μlSty PCR Master Mix。 为了避免污染,每次分配后更换移液管吸头。 每个孔中的总体积为100μl
  15. 用粘合剂膜密封每个反应板
  16. 以高速将每个反应板的中心涡旋3秒
  17. 在560g下旋转板30秒。
  18. 将反应板放在冷却室中的冰上,直到装载到热循环仪上
    将Sty PCR板装载到热循环仪上(阶段3,步骤19-22)
  19. 将板传送到主实验室。
  20. 确保热循环仪盖子已预热。 块应处于室温。
  21. 将每个反应板装在热循环仪上。
  22. 运行GW5.0/6.0 PCR程序。 程序根据您使用的热循环仪而有所不同。 这里使用的实例是GeneAmp PCR系统9700,然而另一个方案可以在制造商的说明 GeneAmp ® PCR系统9700的GW5.0/6.0 PCR程序
    温度
    时间
    循环
    94°C
    3分钟
    1x
    94°C
    30秒
    |
    60°C
    45秒
    30x
    68°C
    15秒
    |
    68°C
    7分钟
    1x
    4°C
    暂停

    卷:
    100微升

    指定最大模式。

    运行凝胶(第3阶段,第23-33步)
    注意:以下说明适用于运行2%TBE凝胶。 为了确保结果一致,从每个PCR中取3μl等分试样。
  23. 当GW5.0/6.0 PCR程序完成后,从热循环仪中取出每个板
  24. 旋转板在560×g 30秒。
  25. 将板置于冰上的冷却室中或保持在4℃
  26. 标记三个新鲜的96孔反应板

    P1Gel,P2Gel 和 P3Gel。
  27. 将3μl2×凝胶加样染料分装到新鲜,标记的PXGel板的A行到D行的每个孔。
  28. 使用12通道P20移液管,将3μl的每个PCR产物从3个Sty PCR板转移到PXGel板的相应板,行和孔。 实施例:将来自P1板上A行每个孔的每个PCR产物的3μl转移到P1Gel平板上A行的相应孔中。
  29. 密封PXGel板。
  30. 旋转每个PXGel板的中心,然后在560×g下旋转30秒。
  31. 将每个PXGel板的每个孔的总体积加载到2%TBE凝胶上
  32. 在120 V下运行凝胶40分钟至1小时。
  33. 验证PCR产物分布在〜200 bp到1,100 bp之间(例如下面的凝胶,图1)

    图1. PCR产物的实施例在2%TBE琼脂糖凝胶上在120V下运行1小时。平均产物分布在约200至1,100bp之间。

阶段4:Nsp限制酶消化
在此阶段,基因组DNA被Nsp I酶消化。

  1. 解冻/将以下试剂放在冰上(约20分钟):
    1. NE缓冲区2
    2. BSA
    3. 基因组DNA(如果冻存)
    4. AccuGENE水
  2. 标记以下管,然后根据需要放置在冷却室或冰上:
    1. 一条12条标有"挖"的管子
    2. 标记为"Dig MM"的2.0ml Eppendorf管
  3. 用基因组DNA制备板,如下:
    1. 以高速旋转板的中心3秒。
    2. 沿560英尺x g 旋转板30秒。
    3. 放回冷却室在冰上。
  4. 准备试剂(除了酶)如下:
    1. 涡旋3次,每次1秒
    2. 脉冲旋转3秒。
    3. 放置在冷却室中。
  5. 预热热循环仪盖,但在室温下离开块。
    注意:将NSP I酶保持在-20°C,直到准备使用。

    准备Nsp摘要主混合(阶段4,步骤6-13)
  6. 将所有试剂和管保持在冰上,通过加入适当体积的来自表4的以下试剂来制备Nsp消化主混合物:
    1. AccuGENE水
    2. NE缓冲区2
    3. BSA
  7. 从冷冻机中取出Nsp I酶,立即放入冷却器中。
  8. 脉冲旋转酶3秒。
  9. 立即将酶添加到主混合物中,然后将剩余的酶放回冷却器中
  10. 高速旋转主混合物3次,每次1秒
  11. 脉冲旋转3秒。
  12. 置于冷却室中。
  13. 将任何剩余的酶返回到冰箱
    表4. Nsp I消解主混合
    试剂
    1样本
    48份样品(15%额外)
    AccuGENE ®
    11.55微升
    637.6微升
    NE缓冲区2(10x)
    2微升
    110.4μl
    BSA(100×; 10mg/ml) 0.2μl
    11微升
    Nsp I(10U /μl)
    1微升
    55.2μl
    总计
    14.75微升
    814.2微升

    将Nsp摘要主混合添加到示例中(第4阶段,第14-21步)
  14. 要将Nsp消化主混合物添加到样品中,使用单通道P200移液管,将67μlNsp消化主混合物分装到标记为Dig的条带管的每个管中。
  15. 使用12通道P20移液管,在冰上的冷却室中,向每个DNA样品中加入14.75μlNsp消化主混合物。 每个孔中的总体积现在为19.75μl
    试剂

    基因组DNA(50 ng /μl)
    5微升
    Nsp消化主混合物
    14.75微升
    总量
    19.75微升
  16. 用胶膜密封板。
  17. 以高速旋转板的中心3秒。
  18. 在560分钟f 或30秒下旋转板。
  19. 确保热循环仪的盖子已预热。
  20. 将板装载到热循环仪上,运行GW5.0/6.0 Digest程序 GW5.0/6.0文摘计划
    温度
    时间
    37°C
    120分钟
    65℃
    20分钟
    4°C
    暂停
  21. 程序完成后,取出版面并以560 x g 旋转30秒。

阶段5:Nsp连接
在此阶段,使用Nsp适配器连接消化的样品。

  1. 解冻/将以下试剂放在冰上(约20分钟):
    1. 适配器Nsp I
    2. T4 DNA连接酶缓冲液(10x)
    3. Nsp消化的样品(如果冷冻)
    4. AccuGENE水
  2. 标记以下管,然后根据需要放置在冷却室或冰上:
    1. 一条12条标有"Lig"的管子
    2. 标记为"Lig MM"的2.0ml Eppendorf管
    3. 解决方案盆地
  3. 按如下方法制备消解样品:
    1. 以高速旋转板的中心3秒。
    2. 沿560英尺x g 旋转板30秒。
    3. 放回冷却室在冰上。
  4. 准备试剂:
    1. 高速涡旋3次,每次1秒(除了酶)
    2. 脉冲旋转3秒。
    3. 放置在冷却室中。
  5. 预热热循环仪盖,但在室温下离开块。

    准备Nsp连接主混合(阶段5,步骤6-12)
  6. 将所有试剂和管保持在冰上,通过向2.0ml Eppendorf管中加入基于表5中的体积的以下试剂来制备Nsp连接主混合物。
    1. 适配器Nsp
    2. T4 DNA连接酶缓冲液(10x)
  7. 从冰箱中取出T4 DNA连接酶,立即置于冰箱中冷藏。
  8. 脉冲旋转T4 DNA连接酶3秒。
  9. 立即将T4 DNA连接酶加入到主混合物中; 然后放回冷却器。
  10.  以高速3次,每次1秒旋转主混音。
  11. 脉冲旋转3秒。
  12.  将主混合物置于冰上。

    表5. Nsp I连接主混合
    试剂
    1样本
    48份样品(15%额外)
    T4 DNA连接酶缓冲液(10x)
    2.5μl
    150μl
    适配器Nsp I(50μM)
    0.75μl
    45微升
    T4 DNA连接酶(400U /μl) 2微升
    120微升
    总计
    5.25微升
    315微升

    将Nsp连接主混合添加到反应中(第5阶段,步骤13-25)
  13. 要将Nsp连接主混合物添加到样品中,使用单通道P100移液管,并在冰上将25μlNsp连接主混合物等分到条管的每个管。
  14. 使用12通道P20移液器,等分5.25微升Nsp连接主混合物在Nsp消化阶段板上的每个反应。 每个孔中的总体积现在为25μl
    试剂

    Nsp消化的DNA
    19.75微升
    Nsp连接主混合物
    5.25微升
    总计
    25μl
  15. 用胶膜密封板。
  16. 以高速旋转板的中心3秒。
  17. 沿560英尺x g 旋转板30秒。
  18. 确保热循环仪盖已预热。
  19. 将板载入热循环仪并运行GW5.0/6.0 Ligate程序。
    GW5.0/6.0升级程序
    温度
    时间
    16°C

    70℃
    20分钟
    4°C
    暂停
  20. 当GW5.0/6.0 Ligate程序完成后,取出板并在560 x g下旋转30秒。
  21. 将板放在冰上的冷却室中。
  22. 稀释每个反应如下:
    1. 将10 ml AccuGENE水倒入溶液池。
    2. 使用12通道P200移液器,为每个反应添加75μl的水 每个孔中的总体积为100μl
      试剂

      Nsp连接的DNA
      25μl
      AccuGENE水
      75微升
      总计
      100微升
  23. 用胶膜密封板。
  24. 以高速旋转板的中心3秒。
  25. 沿560英尺x g 旋转板30秒。
    注意:在PCR之前,用AccuGENE水稀释连接的DNA是至关重要的

阶段6:Nsp PCR
在此阶段,您将等量的每个Nsp连接的样品转移到四个新鲜的96孔板中; 准备Nsp PCR主混合物,并将其加入每个样品; 将每个板放在热循环仪上并运行GW6.0 PCR程序; 并通过在2%TBE凝胶或E-Gel 962%琼脂糖凝胶上运行3μl每种PCR产物来确认PCR。

  1. 解冻/将以下试剂放在冰上(约20分钟):
    1. TITANIUM Taq PCR Buffer
    2. dNTPs
    3. PCR引物002
    4. AccuGENE水
    5. Nsp结合的样品(如果冷冻)
    6. GC-Melt
    7. 解决方案盆地
  2. 标记以下管,然后根据需要放置在冷却室或冰上:
    1. 标记为"P1","P2","P3","P4"的四个96孔反应板
    2. 一个标记为"PCR MM"的50ml Falcon管
  3. 在冰上放置5个板和1个冷却器的足够的冷却室
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bosman, A. (2016). Affymetrix Genome-wide Human SNP Assay. Bio-protocol 6(10): e1806. DOI: 10.21769/BioProtoc.1806.
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