Nov 04, 2024

Public workspaceDNA Library Prep with MGIEasy FS DNA Library Prep Set

DOI
dx.doi.org/10.17504/protocols.io.261gerpkol47/v1
  • 1BGI Research;
  • 2China National GeneBank;
  • 3MGI Tech
Qianyue Ji
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DOI: dx.doi.org/10.17504/protocols.io.261gerpkol47/v1
Protocol Citation: Qianyue Ji, Guoqiang Mai, Xiaohan Wang, Shanshan Liu, Mo Han 2024. DNA Library Prep with MGIEasy FS DNA Library Prep Set. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerpkol47/v1
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
We use this protocol and it's working
Created: October 27, 2024
Last Modified: November 04, 2024
Protocol Integer ID: 110978
Abstract
The MGIEasy FS DNA Library Prep Set(1000006987,MGI Tech) is specifically designed for WGS libraries construction for MGI high-throughput sequencing platform series. This library prep set is optimized to convert 5-400 ng genomic DNA into a customized library. This set uses advanced Adapter Ligation technology and High-fidelity PCR Enzymes to significantly increase library yield and conversion rate. This library prep set is applicable for samples from all common animals, plants, fungus, bacteria, etc., including human, mice, rice, Arabidopsis, yeast, E. coli, Metagenomics.
Materials
Materials MGIEasy Dual Barcode Circularization Kit (Cat. No.: 1000020570) Qubit ssDNA Assay Kit
MGIEasy FS DNA Library Prep Set Supplies: Pipette tips (assorted volumes) 5ml round-bottom polystyrene tubes, PCR tube, centrifuge tube Equipment: Qubit Fluorometer Vortex Micropipettes, various volumes Microcentrifuge Thermocycler Magnetic rack
Fragmentation
Fragmentation
Preparation
Mix the reagents before using and store the remaining reagents immediately after use.
ReagentRequirements
Dilution BufferUser-supplied:place at room temperature (RT).
Frag Buffer IIThaw at RT, mix well, centrifuge briefly, and place on ice.
Frag Enzyme IIKeep on ice.
preparing the reagents

Fragmentation
Add Amount45 µL of genomic DNA to a new 0.2mL PCR tube. Add dilution buffer to make a total volume of Amount45 µL if the fragmentation gDNA volume not enough. Place the tube(s) TemperatureOn ice .
ReagentVolume
gDNAX μL
Dilution buffer45 - X μL
Total45 μL
Normalization of gDNA

Set and run program as table below. The thermocycle will perform the first step reaction described in table below and be kept at Temperature4 °C .
TemperatureTime
70 ℃ Heated lidOn
4℃Hold
30℃8 min
65℃15 min
4℃Hold
Fragmentation reaction conditions

Mix the Frag Enzyme II by inverting 10 times and flicking the bottom gently, ensure that no residual reagent is left at the bottom. Centrifuge briefly and place TemperatureOn ice .
Acorrding to the desired reaction number, prepare the fragmentation mixture in a 0.2mL PCR tube TemperatureOn ice . Mix it well by votexing 3 times, centrifuge briefly, and place TemperatureOn ice .
ReagentVolume per reaction
Frag Buffer II10μL
Frag Enzyme II5 μL
Total15 μL
Fragmentation mixture

Add Amount15 µL of fragmentation mixture to each sample tube. Mix by votexing 3 times, centrifuge briefly, and place TemperatureOn ice .
Make sure the thermocycler has cooled to Temperature4 °C . Place the PCR tube(s) into the thermocycler, and skip the 4℃ Hold step to start the reaction at Temperature30 °C .
PCR
When the program is completed, centrifuge the PCR tube(s) briefly and place TemperatureOn ice .
Double size selection.
The following sample use a Amount36 µL 1st bead selection and a amount Amount12 µL 2nd bead selection to target a 330bp size fragement from fragmented DNA(Amount60 µL ).
Transfer all fragmented DNA to a new 1.5mL centrifuge tube. Add TE buffer for a final volume of Amount60 µL .
Add Amount36 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature forDuration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack forDuration00:05:00 until the liquid is clear. Then, carefully transfer the supernatant to a new 1.5mL centrifuge tube. Retain the supernatant and dicard the beads.
5m
Centrifigation
Add Amount12 µL of DNA Clean Beads to the centrifuge tube withAmount94 µL supernatant. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard all the supernatant.
5m
Centrifigation
While keeping the centrifuge tube on the magnetic rack, add Amount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for 30 sec. Carefully remove and discard the supernatant.
Repeat step 3.8 and try to remove all of the liquid from the tube.
Keep the tube(s) on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube(s) from the magnetic rack and add Amount43 µL of TE to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack forDuration00:05:00 until the liquid is clear. Carefully transferAmount41 µL of supernatant to a new 1.5mL centrifuge tube.
5m
Centrifigation
Quantify the purified size selection products with Qubit dsDNA Assay Kit.
End repair
End repair
End repair
Transfer Amount100 ng DNA sample to a new 0.2mL PCR tube and add TE buffer for a total volume ofAmount40 µL .
Prepare the End repair Mixture TemperatureOn ice .
ReagentVolume per reaction
ERAT Buffer7.1 μL
ERAT Enzyme Mix2.9 μL
Total10 μL
End repair Mixture
Transfer Amount10 µL of the mixture to the 0.2mL PCR tube from step 3. Votex 3 times and centrifuge briefly.
Place the 0.2mL PCR tube into the thermocycler and run the program.
TemperatureTime
70 ℃ Heated lidOn
37 ℃30 min
65℃15 min
4℃Hold
End repair program
PCR
When the program completed, brifly centrifuge to collect the solution at the bottom of the tube.
Adapter ligation
Adapter ligation
Adapter ligation
Add Amount5 µL of adapter to the corresponding sample tube. Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
Prepare the adapter ligation mixture. Vortex it 6 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
ReagentVolume per reaction
Ligation Buffer23.4 μL
DNA Ligase1.6 μL
Total25 μL
adapter ligation mixture
Slowly pipette Amount25 µL of adapter ligation mixture to each sample tube. Vortex it 6 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
The adapter ligation mixture is highly viscous. Pipette slowly and carefully.
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
30 ℃ Heated lidOn
23 ℃30 min
4℃Hold
adapter ligation program
PCR
When the program is completed, centrifuge the PCR tube(s) briefly and place TemperatureOn ice .
Add Amount20 µL TE buffer, for a total volume of Amount100 µL and transfer to a new 1.5mL centrifuge tube.
Cleanup of Adapter-ligated DNA
Cleanup of Adapter-ligated DNA
Cleanup of Adapter-ligated DNA
Add Amount50 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate the sample tube at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the sample tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard all the supernatant.

5m
Centrifigation
Keep the tube on the magnetic rack, add Amount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for 30 sec. Carefully remove and discard the supernatant.
Repeat step 6.4 once. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation.Remove all liquid by using a low-volume pipette.
Keep the tube on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube from the magnetic rack and add Amount21 µL of TE buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully transfer Amount19 µL of supernatant to a new 0.2 mL PCR tube.
5m
PCR Amplification
PCR Amplification
PCR Amplification
Prepare the PCR mixture in a 0.2 mL PCR tube TemperatureOn ice . Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
ReagentVolume per reaction
PCR Enzyme Mix25 μL
PCR Primer Mix6 μL
Total31 μL
PCR mixture
Add Amount31 µL of PCR mixture to each sample tube. Vortex 3 times (3 sec each) and centrifuge briefly to collect the solution at the bottom of the tube.
Place the PCR tube into the thermocycler. Run the program with the following conditions.
TemperatureTimeCycles
TemperatureTimeCycles
105 ℃ Heated lidon
95 ℃3min1
98 ℃20s8
60 ℃15s
72 ℃30s
72 ℃10min1
4 ℃Hold1
PCR program

PCR
When the program is completed, centrifuge the tube(s) briefly and transfer to a new 1.5mL centrifuge tube.
Cleanup of PCR Product
Cleanup of PCR Product
Cleanup of PCR Product
Add Amount50 µL of DNA Clean Beads to each sample tube from 7.4. Mix with a vortexer until all beads are suspended.
Incubate the sample tube at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the sample tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard all the supernatant.
5m
Centrifigation
Keep the tube on the magnetic rack, addAmount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for Duration00:00:30 . Carefully remove and discard the supernatant.
30s
Repeat step 8.4 once. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.
Keep the tube on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube from the magnetic rack and add Amount32 µL of TE buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully transfer Amount30 µL of supernatant to a new 1.5mL centrifuge tube.
5m
Centrifigation
Quality control of PCR product
Quantify the dsDNA with Qubit dsDNA-HS Assay Kit. Yeild for PCR products:>1 pmol.
Assess the size range of purified PCR products with Agilent 2100 Bioanalyzer.

Denaturation and single-stranded circularization
Denaturation and single-stranded circularization
20m 30s
20m 30s
Denaturation
Based on the PCR products concentration, addAmount300 ng of PCR products into a new 0.2 mL PCR tube. If the volume is less than Amount48 µL , add TE Buffer to make a total volumeAmount48 µL .
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
100 ℃ Heated lidOn
95 ℃3 min
4 ℃10 min
Denaturation reaction conditions (Volume: 48 μL)

PCR
After the reaction, centrifuge the tube briefly and place TemperatureOn ice .
Centrifigation
Single-stranded circularization
According to the desired reaction number, prepare the circularization reaction mixture in a new 0.2 mL PCR tube on ice. Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
ReagentVolume per reaction
Dual Barcode Splint Buffer11.6 μL
DNA Rapid Ligase0.5 μL
Total12.1 μL
Circularization reaction mixture

Add Amount12.1 µL of circularization reaction mixture to each sample tube . Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
42 ℃ Heated lidOn
37 ℃10 min
4 ℃Hold
Single-stranded DNA circularization reaction conditions (Volume: 60 μL)

PCR
When the program is completed, place the PCR tube(s) TemperatureOn ice , centrifuge briefly, and immediately proceed to the next step.
Digestion
According to the desired reaction number, prepare the digestion mixture in a 0.2 mL PCR tube TemperatureOn ice . Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
Add Amount4 µL of digestion mixture to each sample tube. Vortex it 3 times (3 sec each), centrifuge briefly, and then place TemperatureOn ice .
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
42 ℃ Heated lidOn
37 ℃10 min
4 ℃Hold
Digestion reaction conditions (Volume: 64 μL)

PCR
When the program is completed, centrifuge the tube briefly and immediately add Amount7.5 µL of Digestion Stop Buffer to each sample tube. Vortex it 3 times (3 sec each), centrifuge briefly, and place TemperatureOn ice .
Cleanup of digestion product
Mix the DNA Clean Beads thoroughly. Add Amount130 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard the supernatant.
5m
While keeping the tube(s) on the magnetic rack, add Amount160 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for Duration00:00:30 . Carefully remove and discard the supernatant.
30s
Repeat step 12.4. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.
Keep the tube(s) on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube(s) from the magnetic rack and add Amount25 µL of TE Buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully transfer Amount24 µL of supernatant to a new 1.5 mL centrifuge tube.
5m
Centrifigation
QC of digestion product
Quantify the ssCir with Qubit ssDNA Assay Kit. The final Enzymatic Digestion products (ssDNA,
ng) / input products of PCR (dsDNA, 300 ng) should be ≥ 7%.