Sep 14, 2015
NEXTflex™ mtDNA-Seq for blood samples
External link: http://www.biooscientific.com/Portals/0/Manuals/NGS/5280-01-NEXTflex-mtDNA-Seq-Kit.pdf
Protocol Citation: Bioo Scientific: NEXTflex™ mtDNA-Seq for blood samples. protocols.io https://dx.doi.org/10.17504/protocols.io.dqy5xv
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 03, 2015
Last Modified: March 23, 2018
Protocol Integer ID: 1528
Abstract
The NEXTflex™ mtDNA-Seq Kit is designed to prepare single or paired-end DNA libraries of mitochondrial DNA (mtDNA) for sequencing using Illumina® platforms. The procedure isolates mitochondrial DNA from genomic DNA (gDNA) by selective digestion of linear nuclear DNA, followed by fragmentation of mtDNA and library preparation.
Guidelines
Contents, Storage and Shelf Life
The NEXTflex⢠mtDNA-Seq Kit contains enough material to isolate mtDNA and prepare 8 libraries for Illuminaî compatible sequencing. The shelf life of all reagents is 12 months when stored properly. 6X Loading Dye, Nuclease-free Water, and Resuspension Buffer can be stored at room temperature. All other components should be safely stored at -20ðC.
Required Materials Not Provided
⢠2 - 8 üg of genomic DNA in up to 35 üL nuclease-free water
⢠(Optional)NEXTflex⢠ChIP-Seq Barcodes â 6 , 12 , 24 , 48 or NEXTflex-96⢠ChIP-Seq Barcodes (Cat # 514120, 514121, 514122, 514123, 514124)
⢠Ethanol 100% (room temperature)
⢠Covaris System (S2, E210) or other device for fragmenting DNA
⢠96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
⢠96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
⢠Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
⢠Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
⢠Magnetic Stand-96 (Life Technologies, Cat # AM10027) or similar
⢠Heat block at 37ðC and at 70ðC
⢠Thermocycler
⢠2, 10, 20, 200 and 1000 üL pipettes, multichannel pipettes
⢠Nuclease-free barrier pipette tips
⢠Microcentrifuge
⢠1.5 mL nuclease-free microcentrifuge tubes
⢠Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65úC (Boston Bioproducts, Cat # P-730)
⢠1X TAE buffer
⢠SYBR Gold (Invitrogen, Cat # S11494)
⢠UV transilluminator or gel documentation instrument
⢠Gel electrophoresis apparatus
⢠Electrophoresis power supply
⢠Vortex
⢠MiVac (SpeedVac)
NEXTflex⢠mtDNA-Seq Flow Chart
Figure 1: Sample flow chart with approximate times necessary for each step.
Starting Material
The NEXTflex⢠mtDNA-Seq Kit has been optimized and validated using 2 â 8 üg of genomic DNA isolated either from human blood or cell samples. This kit provides sufficient reagents for 8 nuclear DNA digestions and mtDNA library preparations.
It is recommended that the mtDNA isolation test (see "Optional Quality Control of Nuclear DNA Digest" section) is performed with each isolation. Primers and PCR master mix for this test are provided with the kit. Undigested gDNA may be used as a control (not provided).
Reagent Preparation
1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each tube prior to use.
2. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use.
Figure 3A:ÃÂ Bioanalyzer Gel Image
Figure 3 B: Bioanalyzer Electropherogram Image
Figure 3A & 3B: Example of a mtDNA Library Size Distribution. Upon isolation the mtDNA was sheared to 150-200 bp, a total of 18 cycles of PCR were performed. 1 üL of the resulting library was run on an Agilent High Sensitivity DNA chip to verify size. Using a Qubitî 2.0 Fluorometer & Qubitî dsDNA HS Assay Kit, the concentration of the library was determined to be > 10 nM.
Materials
MATERIALS
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Magnetic Stand -96Life TechnologiesCatalog #AM10027
96 well Library Storage and Pooling PlateFisher ScientificCatalog #AB-0765
Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ºCBoston BioproductsCatalog #P-730
SYBR GoldThermo ScientificCatalog #S-11494
STEP MATERIALS
Agencourt AMPure XPBeckman CoulterCatalog #A63880
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
SYBR GoldThermo ScientificCatalog #S-11494
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
SYBR GoldThermo ScientificCatalog #S-11494
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Protocol materials
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
96 well Library Storage and Pooling PlateFisher ScientificCatalog #AB-0765
Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ºCBoston BioproductsCatalog #P-730
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
SYBR GoldThermo ScientificCatalog #S-11494
Magnetic Stand -96Life TechnologiesCatalog #AM10027
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
SYBR GoldThermo ScientificCatalog #S-11494
SYBR GoldThermo ScientificCatalog #S-11494
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
SYBR GoldThermo ScientificCatalog #S-11494
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Safety warnings
Bioo Scientific strongly recommends that you read the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or Bioo Scientific at nextgen@biooscientific.com.
• Do not use the kit past the expiration date.
• DTT in buffers may precipitate after freezing. If precipitate is seen, vortex buffer for 1-2 minutes or until the precipitate is in solution. The performance of the buffer is not affected once precipitate is in solution.
• Ensure pipettes are properly calibrated as library preparations are highly sensitive to pipetting error.
• Do not heat the DNA Adapters above room temperature.
• Try to maintain a laboratory temperature of 20°–25°C (68°–77°F).
• DNA sample quality may vary between preparations. It is the user’s responsibility to utilize high quality DNA. DNA that is heavily nicked or damaged may cause library preparation failure. Absorbance measurements at 260 nm are commonly used to quantify DNA and 260 nm / 280 nm ratios of 1.8 - 2.0 usually indicate relatively pure DNA. Other quantification methods using fluorescent dyes may also be used. The user should be aware that contaminating RNA, nucleotides and single-stranded DNA may affect the amount of usable DNA in a sample preparation.
• Vortex the genomic DNA for 10 seconds before quantification. It is critical that the user quantifies the input gDNA correctly. The yield and purity of isolated mtDNA is dependent on the concentration of gDNA used.
Before start
Reagent Preparation
1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each tube prior to use.
2. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use.
Reagent Preparation
Reagent Preparation
Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each tube prior to use.
Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use.
Selective Digestion of Nuclear DNA (Blood Samples)
Selective Digestion of Nuclear DNA (Blood Samples)
For each sample, combine the following reagents on ice in nuclease-free microcentrifuge tubes:
| _ μL | Nuclease-free Water | |
| _ μL | gDNA (2 – 4 μg) | |
| 5 μL | NEXTflex™ mtDNA Buffer Mix 1 | |
| 5 μL | NEXTflex™ mtDNA Buffer Mix 2 | |
| 5 μL | NEXTflex™ Nuclear DNA Digest Mix | |
| 50 μL | TOTAL |
Note
Materials
Bioo Scientific Supplied
BLUE CAP - NEXTflex™ mtDNA Buffer Mix 1, NEXTflex™ mtDNA Buffer Mix 2, NEXTflex™ Nuclear DNA Digest Mix
WHITE CAP - Nuclease-free Water
User Supplied
gDNA isolated from blood in up to 35 μL nuclease-free water
Heat block
1.5 mL Microcentrifuge tubes
Microcentrifuge
Ice
Agencourt AMPure XP Magnetic Beads (room temperature)
80% Ethanol, freshly prepared (room temperature)
Magnetic rack
Mix well by pipetting.
Incubate in a heat block for 48 hours at 37°C.
48:00:00
Incubate the sample at 70°C for 30 minutes.
00:30:00
Spin the tubes for 10 seconds to collect contents of the tube.
00:00:10
Add 50 μL of AMPure XP Beads to each sample and mix well by pipetting.
50 µL
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate at room temperature for 5 minutes.
00:05:00
Place the tube on the magnetic rack at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in the tube.
Wash #1: With tube on rack, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With tube on rack, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
00:00:30
Remove the tube from the magnetic rack and let dry at room temperature for 3 minutes. Do not overdry the beads.
00:03:00
Resuspend dried beads with 36 μL Nuclease-free Water. Mix well by pipetting. Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place tube on magnetic rack for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 35 μL of clear sample to a fresh microcentrifuge tube.
To ensure complete removal of nuclear DNA contamination, repeat the digestion with 35 μL eluted material from Step 18. Set up the reaction as follows.
| 35 μL | Eluted DNA (Step 18) | |
| 5 μL | NEXTflex™ mtDNA Buffer Mix 1 | |
| 5μL | NEXTflex™ mtDNA Buffer Mix 2 | |
| 5 μL | NEXTflex™ Nuclear DNA Digest Mix | |
| 50 μL | TOTAL |
Incubate for 24 hours at 37°C.
24:00:00
Repeat steps 6-18.
Note
If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20 °C. To continue, thaw your frozen samples on ice before proceeding to section "Optional Quality Control of Nuclear DNA Digest".
Section "Optional Quality Control of Nuclear DNA Digest" is an optional quality control check of nuclear DNA digestion. Primers and PCR master mix for this test are provided with this kit. Performing this test with undigested gDNA as a control is highly recommended.
If not performing the section test, proceed to section "Fragmentation of mtDNA" for Fragmentation of mtDNA.
Optional Quality Control of Nuclear DNA Digest
Optional Quality Control of Nuclear DNA Digest
For each sample prepare two separate reactions in adjacent wells of a 96-well PCR Plate on ice as described below:
Note
Materials
Bioo Scientific Supplied
BROWN CAP - NEXTflex™ mtDNA Primer Mix, NEXTflex™ Nuclear DNA Primer Mix
GREEN CAP - NEXTflex™ PCR Master Mix WHITE CAP - 6X Loading Dye, MW 100 bp Ready-to-Load Ladder
WHITE CAP - Nuclease-free Water
User Supplied
2 μL of isolated mtDNA (from sections "Selective Digestion of Nuclear DNA (Cell Samples)" and " Selective Digestion of Nuclear DNA (Blood Samples)")
Thermocycler
2% Agarose gel
Electrophoresis system
96 well PCR Plate Non-skirted Phenix ResearchCatalog #MPS-499
Mix well by pipetting.
Apply adhesive PCR plate seal and place in thermocycler for the following PCR cycles:
Prepare pre-stained SYBR Gold 2% or Ethidium Bromide TAE agarose gel. Mix and pour into gel tray. Load 3 μL of the PCR product mixed with 2 μL of 6X Loading Dye and 7 μL of Nuclease-free Water. Load the samples on the gel along with 6 μL of MW 100 bp Ready-to-Load Ladder.
SYBR GoldThermo ScientificCatalog #S-11494
Run the gel with 1X TAE buffer at 100-120V for 60-120 minutes, or until bands have adequately resolved.
01:00:00
Visualize the gel on UV transilluminator or gel documentation instrument. Check for the presence of a mtDNA band and absence/significant reduction (compared to the control) of nuclear DNA bands to proceed with the subsequent steps (Fig. 2 in Guidelines).
Fragmentation of mtDNA
Fragmentation of mtDNA
Adjust mtDNA sample volume to 130 μL with Nuclease-free Water. Set pipette to 130 μL and mix well by pipetting.
Note
Materials
Bioo Scientific Supplied
WHITE CAP - Nuclease-free Water
User Supplied
Up to 40 μL isolated mtDNA (from STEP A1/A2)
Covaris AFA microTUBE
Covaris Focused-Ultrasonicator System
Vacuum concentrator
Qubit® fluorometer
Transfer the sample to a Covaris tube. Follow manufacturer's instructions. For example, using the Covaris S2 system, the following parameters will produce fragments of 150-200 bp
Peak Intensity – 5
Duty cycle – 10%
Cycles per burst – 200
Time – 180 s
Transfer your sample from a Covaris tube to a 15 mL microcentrifuge tube.
Option 1: SpeedVac - following sonication, spin down sample in a SpeedVac for 2 hours at 45°C to reduce the volume of your sample to ≤ 40 μL. Do not let the sample dry down completely.
02:00:00
Option 2: Bead Cleanup – Alternatively a 2X AMPure XP bead clean up can be done as follows:
Protocol
CREATED BY
Bioo Scientific
Add 260 μL of AMPure XP beads to each sample Mix thoroughly.
250 µL
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate sample at room temperature for 5 minutes.
00:05:00
Place the tube on the magnetic rack at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in the tube.
Wash #1: With the tubes on the rack, gently add 500 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With the tubes on the rack, gently add 500 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
Remove the tube from the magnetic rack and let dry at room temperature for 3 minutes. Do not overdry the beads.
00:03:00
Resuspend the dried beads with 42 μL Nuclease-free Water. Mix well by pipetting. Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place the tube on magnetic rack for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 40 μL of clear sample to a fresh microcentrifuge tube.
After concentrating the sample, Qubit® dsDNA reagents can be used to quantify the DNA concentration.
Note
Note that mtDNA accounts for 0.01% to 1% of total DNA and the concentration of isolated mtDNA can be very low. Concentrations typically vary between 0.1 to 1 ng/μL. It is recommended to use all of the fragmented mtDNA in library prep.
End Repair
End Repair
For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate:
| 40 μL | mtDNA (from section "Fragmentation of mtDNA") | |
| 7 μL | NEXTflex™ mtDNA End Repair Buffer Mix | |
| 3 μL | NEXTflex™ mtDNA End Repair Enzyme Mix | |
| 50 μL | TOTAL |
Note
Materials
Bioo Scientific Supplied
CLEAR CAP - NEXTflex™ mtDNA End Repair Buffer Mix, NEXTflex™ mtDNA End Repair Enzyme Mix
WHITE CAP - Nuclease-free Water
User Supplied
mtDNA in 40 μL Nuclease-free Water (from section "Fragmentation of mtDNA")
96 well PCR Plate
Adhesive PCR Plate Seal
Microcentrifuge
Ice
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 22°C.
00:30:00
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Clean-Up
Clean-Up
Add 80 μL of AMPure XP Beads to each sample and mix well by pipetting.
80 µL
Note
Materials
Bioo Scientific Supplied
WHITE CAP - Resuspension Buffer
User Supplied
Agencourt AMPure XP Magnetic Beads (room temperature)
80% Ethanol, freshly prepared (room temperature)
Magnetic Stand
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate sample at room temperature for 5 minutes.
00:05:00
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. Note: Do not overdry the beads.
00:03:00
Resuspend dried beads with 17 μL Resuspension Buffer. Mix well by pipetting. Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 16 μL of clear sample to a new well.
Note
If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20°C. To restart, always thaw your frozen samples on ice before proceeding to section "3’ Adenylation".
3’ Adenylation
3’ Adenylation
Combine the following in the 96 well PCR Plate:
| 16 μL | End-Repaired DNA (from section "Clean-Up") | |
| 4.5 μL | NEXTflex™ mtDNA Adenylation Mix | |
| 20.5 μL | TOTAL |
Note
Materials
Bioo Scientific Supplied
RED CAP - NEXTflex™ mtDNA-Seq Adenylation Mix
User Supplied
Thermocycler (set to 37°C)
16 μL of End Repaired DNA (from section "Clean-Up")
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 37°C.
00:30:00
Adapter Ligation
Adapter Ligation
For each sample, combine the following reagents (in this order) in the 96-well PCR Plate:
| 20.5 μL | 3’ Adenylated DNA (from the above section) | |
| 27.5 μL | NEXTflex™ mtDNA Ligation Mix | |
| 2.0 μL | NEXTflex™ mtDNA Adapter or NEXTflex™ ChIP Barcode | |
| 50 μL | TOTAL |
Note
Materials
Bioo Scientific Supplied
PURPLE CAP - NEXTflex™ mtDNA Ligation Mix (Thaw right before use and store immediately after use at -20°C), NEXTflex™ ChIP Adapter
User Supplied
20.5 μL 3’ Adenylated DNA (from section "3’ Adenylation")
(Optional) NEXTflex™ ChIP Barcodes – 6 / 12 / 24 / 48 / 96 (Cat # 514120, 514121, 514122, 514123, 514124)
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 15 minutes at 22°C.
00:15:00
Adhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
Clean-Up 2
Clean-Up 2
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
40 µL
Note
Materials
Bioo Scientific Supplied
WHITE CAP - Resuspension Buffer
User Supplied
Agencourt AMPure XP Magnetic Beads (room temperature)
80% Ethanol, freshly prepared (room temperature)
Magnetic Stand
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate at room temperature for 5 minutes.
00:05:00
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturb beads. Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
00:00:30
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
00:03:00
Note
Do not overdry the beads.
Resuspend dried beads with 52 μL Resuspension Buffer. Mix well by pipetting and ensuring beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 50 μL of clear sample to new well.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
40 µL
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate at room temperature for 5 minutes.
00:05:00
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes oruntil the supernatant appears clear.
00:05:00
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturb beads. Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
00:00:30
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
00:03:00
Note
Do not overdry the beads.
Resuspend dried beads with 38 μL Resuspension Buffer Mix well by pipetting Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 36 μL of clear sample to new well.
Note
If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20°C. To restart, always thaw your frozen samples on ice before proceeding to section 'PCR Amplification'.
PCR Amplification
PCR Amplification
For each sample, combine the following reagents on ice in the 96 well PCR plate:
| 36 μL | Purified Ligation Product (from section "Clean-Up 2") | |
| 12 μL | NEXTflex™ PCR Master Mix | |
| 2 μL | NEXTflex™ Primer Mix | |
| 50 μL | TOTAL |
Note
Materials
Bioo Scientific Supplied
GREEN CAP - NEXTflex™ Primer Mix , NEXTflex™ PCR Master Mix
WHITE CAP - Resuspension Buffer
User Supplied
Thermocycler
96 Well PCR Plate
Agencourt AMPure XP Magnetic Beads (room temperature)
80% Ethanol, freshly prepared (room temperature)
Magnetic Stand
*36 μL Ligation Product (from section "Clean-Up 2")
Mix well by pipetting.
PCR Cycles:
Note
PCR cycles will vary depending on the amount of starting material and quality of your sample. Further optimization may be necessary. Always use the least number of cycles possible.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
40 µL
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate at room temperature for 5 minutes.
00:05:00
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturb beads. Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
00:00:30
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
00:03:00
Note
Note: Do not overdry the beads.
Resuspend dried beads with 52 μL Resuspension Buffer. Mix well by pipetting and ensuring beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 50 μL of clear sample to new well.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
40 µL
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Incubate at room temperature for 5 minutes.
00:05:00
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
00:05:00
Set pipette to 90 μL and gently remove clear supernatant taking care not to disturb beads. Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette.
00:00:30
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. Ensure all ethanol has been removed.
00:00:30
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
00:03:00
Note
Do not overdry the beads.
Note
Do not overdry the beads.
Resuspend dried beads with 16 μL Resuspension Buffer. Mix well by pipetting. Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
00:02:00
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
00:05:00
Gently transfer 15 μL of clear sample to a well of a new 96 well PCR Plate.
qPCR is recommended to quantitate DNA library templates for optimal cluster density.