NEXTflex™ mtDNA - Seq for cell samples
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 .
For each sample , combine the following reagents on ice in nuclease - free microcentrifuge tubes :
μLNuclease - free Water _ μLgDNA ( 4 – 8 μg ) 5 μLNEXTflex™ mtDNA Buffer Mix 15 μLNEXTflex™ mtDNA Buffer Mix 25 μLNEXTflex™ Nuclear DNA Digest Mix50 μLTOTAL .
Mix well by pipetting .
Incubate in a heat block for 48 hours at 37°C .
Incubate the sample at 70°C for 30 minutes .
Spin the tube for 10 seconds to collect contents of the tube .
Add 50 μL of AMPure XP Beads to each sample and mix well by pipetting .
Incubate at room temperature for 5 minutes .
Place the tube on the magnetic rack at room temperature for 5 minutes or until thesupernatant appears clear .
Remove and discard clear supernatant taking care not to disturb beads .
Some liquidmay 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 .
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 .
Remove the tube from the magnetic rack and let dry at room temperature for 3 minutes .
Do not overdry the beads .
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 .
Place tube on magnetic rack for 5 minutes or until the sample appears clear .
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 of eluted material from Step 18 .
Set up the reaction as follows :
35 μLEluted DNA ( Step 18 ) 5 μLNEXTflex™ mtDNA Buffer Mix 15 μLNEXTflex™ mtDNA Buffer Mix 25 μLNEXTflex™ nuclear DNA Digest Mix50 μLTOTAL .
Incubate for 2 hours at 37°C .
Repeat steps 6 - 18 .
For each sample prepare two separate reactions in adjacent wells of a 96 - well PCR Plate on ice as described below :
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 .
Run the gel with 1X TAE buffer at 100 - 120V for 60 - 120 minutes , or until bands have adequately resolved .
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 ) .
Adjust mtDNA sample volume to 130 μL with Nuclease - free Water .
Set pipette to 130 μL and mix well by pipetting .
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 hoursat 45°C to reduce the volume of your sample to ≤ 40 μL .
Do not let the sample drydown completely .
Option 2 : Bead Cleanup – Alternatively a 2X AMPure XP bead clean up can be done as follows :
After concentrating the sample , Qubit® dsDNA reagents can be used to quantify the DNA concentration .
For each sample , combine the following reagents on ice in a nuclease - free 96 well PCR Plate :
40 μLmtDNA ( from section " Fragmentation of mtDNA " ) 7 μLNEXTflex™ mtDNA End Repair Buffer Mix3 μLNEXTflex™ mtDNA End Repair Enzyme Mix50 μLTOTAL .
Mix well by pipetting .
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 22°C .
Add 80 μL of AMPure XP Beads to each sample and mix well by pipetting .
Incubate sample at room temperature for 5 minutes .
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear .
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 .
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 .
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 .
Place plate on magnetic stand for 5 minutes or until the sample appears clear .
Gently transfer 16 μL of clear sample to a new well .
Combine the following in the 96 well PCR Plate :
16 μLEnd - Repaired DNA ( from section " Clean - Up " ) 4.5 μLNEXTflex™ mtDNA Adenylation Mix20.5 μLTOTAL .
Mix well by pipetting .
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 37°C .
For each sample , combine the following reagents ( in this order ) in the 96 - well PCR Plate :
20.5 μL3’ Adenylated DNA ( from the above section ) 27.5 μLNEXTflex™ mtDNA Ligation Mix2.0 μLNEXTflex™ mtDNA Adapter or NEXTflex™ ChIP Barcode50 μLTOTAL .
Mix well by pipetting .
Apply adhesive PCR plate seal and incubate on a thermocycler for 15 minutes at 22°C .
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting .
Incubate at room temperature for 5 minutes .
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear .
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturbbeads .
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 .
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 .
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes .
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 .
Place plate on magnetic stand for 5 minutes or until the sample appears clear .
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 .
Incubate at room temperature for 5 minutes .
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear .
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 .
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 .
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes .
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 .
Place plate on magnetic stand for 5 minutes or until the sample appears clear .
Gently transfer 36 μL of clear sample to new well .
For each sample , combine the following reagents on ice in the 96 well PCR plate :
36 μLPurified Ligation Product ( from section " Clean - Up 2 " ) 12 μLNEXTflex™ PCR Master Mix2 μLNEXTflex™ Primer Mix50 μLTOTAL .
Mix well by pipetting .
PCR Cycles : Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting .
Incubate at room temperature for 5 minutes .
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutesor until the supernatant appears clear .
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturbbeads .
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 .
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 .
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes .
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 .
Place plate on magnetic stand for 5 minutes or until the sample appears clear .
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 .
Incubate at room temperature for 5 minutes .
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutesor until the supernatant appears clear .
Set pipette to 90 μL and gently remove clear supernatant taking care not to disturbbeads .
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 .
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 .
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes .
Resuspend dried beads with 16 μL Resuspension Buffer .
Mix well by pipetting .
Ensurebeads are no longer attached to the side of the well .
Incubate resuspended beads at room temperature for 2 minutes .
Place plate on magnetic stand for 5 minutes or until the sample appears clear .
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 .
