manual NEBNext DNA Library Prep Master Mix Set for Illumina
Contents
1. Si gn up for the NEBNext e newsletter m m E Scan this code or visit www neb com NEBNextnews2 to sign up for the NEBNext bimonthly e newsletter to learn about new NEBNext products recent publications and advances in library prep for next gen sequencing Regist Quali AMPU Management Management ISO 9001 ISO 14001 ISO 13485 ered Registered Registered lity Environmental Medical Devices USER is protected by U S Patent No 7 435 572 New England Biolabs Inc NEW ENGLAND BIOLABS NEBNEXT and Q5 are registered trademarks of New England Biolabs Inc LITMUS USER and ULTRA are trademarks of New England Biolabs Inc RE is a registered trademark of Beckman Coulter Inc E GEL is a registered trademark of Life Technologies Inc BIOAN ALYZER is a registered trademark of Agilent Technologies Inc ILLUMINA is a registered trademark of Illumina Inc LOBIN D is a registered trademark of Eppendorf AG MILLI Q is a registered trademark of Millipore Corporation QIAQUICK is a registered trademark of Qiagen This product is intended for research purposes only This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals Cloned at Recombinant Optimum Requires Heat NEBiolabs Ri Enzyme Buffer BSA eel Inactivation Table of Contents ADDIICAN ONS eneinio 13 Sahl tedicness ease 2 PROTOC2. Total library size insert adaptor Bead DNA ratio Ist bead selection Bead DNA ratio 0 2X 0 2X 2nd bead selection 0 2X 0 2X 0 15X 0 15X 0 15X Table 1 1 Recommended conditions for dual bead based size selection The following size selection protocol is for libraries with 200 bp inserts only For libraries with different size fragment inserts please optimize bead DNA ratio according to Table 1 1 above Note X refers to the original sample volume of 100 pl Add 80 ul 0 8X resuspended AMPure XP Beads to 100 ul DNA solution Mix well on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature Place the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully transfer the supernatant to a new tube well Caution do not discard the supernatant Discard beads that contain the large fragments Add 20 ul 0 2X resuspended AMPure XP Beads to the supernatant mix well and incubate for 5 minutes at room temperature Put the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully remove and discard the supernatant Be careful not to disturb the beads that contain DNA targets Caution do not discard beads Add 200 ul of freshly prepared 80 ethanol to the tube PCR plate while in the magnetic stand In
3. Follow Section 1 8C if you are using the following oligos 25 pM primer NEBNext Singleplex Oligos for Illumina NEB E7350 lots 0051402 or 0061410 NEBNext Multiplex Oligos for Illumina Set 1 NEB E7335 lots 0071402 or0081407 NEBNext Multiplex Oligos for Illumina Set 2 NEB E7500 lots 0051402 or 0061407 1 8A PCR Enrichment of Adaptor Ligated DNA 1 Mix the following components in sterile strip tubes Adaptor Ligated DNA Fragments 15 ul blue Index Primer i7 Primer 5 yl blue Universal PCR Primer i5 Primer 5 ul blue NEBNext Q5 Hot Start HiFi PCR Master Mix 25 ul Total volume 50 ul The primers are provided in NEBNext Singleplex NEB 7350 or Multiplex NEB E7335 E7500 E7600 Oligos for Illumina For use with Dual Index Primers NEB E7600 look at the NEB 7600 manual for valid barcode combinations and tips for setting up PCR reactions For use with NEBNext Multiplex Oligos NEB 7335 or E7500 use only one Index Primer per PCR reaction For use with Dual Index Primers NEB E7600 use only one i7 Primer per reaction For use with Dual Index Primers NEB E7600 use only one i5 Primer per reaction 2 PCR cycling conditions 0 0 B NS TEMP TIME CYCLES Initial Denaturation 98 C 30 seconds 1 Denaturation 98 C 10 seconds 2 4 Annealing Extension 65 C 75 seconds Final Extension 65 C 5 minutes 1 Hold 4 C o0 If library construction was performed with 5
4. by agarose gel electrophoresis Endonuclease Activity Incubation of a minimum of 50 units of this enzyme with 1 ug of X174 RF DNA in assay buffer for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis Phosphatase Activity Incubation of a minimum of 50 units of this enzyme in protein phosphatase assay buffer 1 M diethanolamine pH 9 8 and 0 5 mM MgCI containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenyl ene anion as determined by spectrophotometric analysis at 405 nm RNase Activity Incubation of a minimum of 5 units of this enzyme with 40 ng of a FAM labeled RNA transcript for 16 hours at 37 C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis Exonuclease Activity Incubation of a minimum of 200 units of this enzyme with 1 ug sonicated H DNA 10 cpm pg for 4 hours at 37 C in 50 ul reaction buffer releases lt 0 1 radioactivity 3 5 Exonuclease Activity Incubation of a minimum of 50 units of enzyme in 20 ul of a 10 nM solution of a fluorescent 5 FAM labeled oligonucleotide for 30 minutes at 37 C yields no detectable 3 5 degradation as determined by capillary electrophoresis Functional Activity Nucleotide Incorporation One unit of this enzyme incorporates 10 nmol of dNTP into acid precipitable material in a total reaction volume of 50 ul in 30 minute
5. diethanolamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm Lot Controlled 14 Klenow Fragment 3 5 exo E6044A 0 036 ml E6044AA 0 180 ml De he Store at 20 C Description Klenow Fragment 3 5 exo is an N terminal truncation of DNA Polymerase which retains polymerase activity but lacks 5 3 exonuclease activity Mutations D355A E357A abolish the 3 5 exonuclease activity 1 Klenow Fragment 3 5 exo with dA Tailing buffer can be used to add a dAMP to the 3 end at a blunt DNA fragment 2 Source An coli strain containing a plasmid with a fragment of the E coli polA D355A E357A gene starting at codon 324 Supplied in 25 mM Tris HCl pH 7 4 0 1 mM EDTA 1 mM DTT and 50 glycerol Quality Control Assays SDS PAGE Purity SDS PAGE analysis of this enzyme indicates gt 95 enzyme purity 16 Hour Incubation 50 yl reactions containing a minimum of 5 units of this enzyme and 1 ug of HindIII digested Lambda DNA incubated for 16 hours at 37 C results in no de tectable non specific nuclease degradation as determined by agarose gel electrophore sis 50 pl reactions containing a minimum of 5 units of this enzyme and 1 pg T3 DNA incubated for 16 hours at 37 C also results in no detectable non specific nuclease degradation as determined
6. yg of starting material use 2 3 cycles of amplification If starting material was 1 ug use 4 cycles of amplification However optimization of PCR cycle number may be required to avoid over amplification 3 Proceed to Cleanup Using Ampure XP Beads in Section 1 9 1 8B PCR Enrichment of Adaptor Ligated DNA 1 3 Mix the following components in sterile strip tubes Adaptor Ligated DNA Fragments 15 ul blue Index Universal Primer Mix 10 pl blue NEBNext Q5 Hot Start HiFi PCR Master Mix 25 ul Total volume 50 ul The primers are provided in NEBNext Multiplex Oligos for Illumina NEB E6609 Please refer to the NEB E6609 manual for valid barcode cobinations and tips for setting up PCR reactions PCR cycling conditions GYCLE STEP TEMP TIME CYCLES Initial Denaturation 98 C 30 seconds 1 Denaturation 98 C 10 seconds 2 4 Annealing Extension 65 C 75 seconds Final Extension 65 C 5 minutes 1 Hold 4 C o0 If library construction was performed with 5 pg of starting material use 2 3 cycles of amplification If starting material was 1 ug use 4 cycles of amplification However optimization of PCR cycle number may be required to avoid over amplification Proceed to Cleanup Using Ampure XP Beads in Section 1 9 1 8C PCR Enrichment of Adaptor Ligated DNA 1 Mix the following components in sterile strip tubes Adaptor Ligated DNA Fragments 15 ul blue Index Primer 2 5 ul blue Univ
7. 80 ethanol to the tube PCR plate while in the magnetic stand Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant Repeat Step 5 once Air dry the beads for 5 minutes while the tube PCR plate is on the mag netic stand with the lid open Caution Do not overdry the beads This may result in lower recovery of DNA target Remove the tube plate from the magnet Elute the DNA target from the beads by adding 30 ul of 0 1X TE Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature Put the tube PCR plate in the magnetic stand until the solution is clear Without disturbing the bead pellet carefully transfer 25 ul of the super natant to a clean LoBind Eppendorf AG tube Libraries can be stored at 20 C Dilute 2 3 pl of the library 20 fold with 10 mM Tris HCl or 0 1X TE and assess the library quality on a Bioanalyzer Agilent Technologies Inc high sensitivity chip Check that the electropherogram shows a narrow distribution with a peak size approximately 300 320 bp Figure 1 1 Example of DNA library size distribution on a Bioanalyzer 35 100 200 300 400 600 1 0002 000 10 380 NEBNext End Repair Enzyme Mix E6041A 0 06 ml E6041AA 0 3 ml DE Store at 20 C Description NEBNext End Repair Enzyme Mix is optimized to convert 1 to 5 ug of fragmented DNA to repaired DNA having 5 phosphorylated blunt en
8. OLS sesi asieins treatise eae ERE EA E salads Hed son edangieaieales 3 NEBNext End Repair Enzyme Mix 0 0 00 0 0 00 0 12 NEBNext End Repair Reaction Buffer 13 Klenow Fragment 3 35 eX0 te ienn caedes de raven n A a ents 14 NEBNext dA Tailing Reaction Buffer 0 0 0 00 00 cece 15 Quick T4 DNA HG ASO ic csese ne delivdaanesdiesnabatendu a o 16 NEBNext Quick Ligation Reaction Buffer 0 0 0 0 0 eee 17 NEBNext Q5 Hot Start HiFi PCR Master M X 18 Revision History aaaea c ett ee teens 19 The Library Kit Includes The volumes provided are sufficient for preparation of up to 12 reactions NEB 6040S and 60 reactions NEB E6040L All reagents should be stored at 20 C green NEBNext End Repair Enzyme Mix green NEBNext End Repair Reaction Buffer 10X yellow Klenow Fragment 3 5 exo iN yellow NEBNext dA Tailing Reaction Buffer 10X red Quick T4 DNA Ligase red NEBNext Quick Ligation Reaction Buffer 5X blue NEBNext Q5 Hot Start HiFi PCR Master Mix Required Materials Not Included 80 Ethanol freshly prepared Nuclease free Water 0 1X TE pH 8 0 10 mM Tris HCI pH 7 5 8 0 optional DNA LoBind Tubes Eppendorf 02243 1021 AMPure XP Beads Beckman Coulter Inc A6388 1 NEBNext Singleplex or Multiplex Oligos for Illumina 7350 7335 E7500 or E7600 Magnetic rack stand PCR Machine Applications The NEBNext DNA Librar
9. X End Repair Buffer as determined by capillary electrophoresis Lot Controlled NEBNext End Repair Reaction Buffer E6042A 0 120 mi Concentration 10X E6042AA 0 6 mi Store at 20 C 1X NEBNext End Repair Reaction Buffer 50 mM Tris HCl 10 mM MgCl 10 mM DTT 1 mM ATP 0 4 mM dATP 0 4 mM dCTP 0 4 mM dGTP 0 4 mM dTTP pH 7 5 25 C Quality Control Assays 16 Hour Incubation 50 ul reactions containing this reaction buffer at 1X concentration and 1 Wg of Hindlll digested Lambda DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis 50 ul reactions containing this reaction buffer at 1X concentration and 1 pg T3 DNA incubated for 16 hours at 37 C also results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis Endonuclease Activity Incubation of this reaction buffer at a 1X concentration with 1 ug of oX174 RF DNA for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis RNase Activity Incubation of this reaction buffer at 1X concentration with 40 ng of a FAM labeled RNA transcript for 16 hours at 37 C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis Phosphatase Activity Incubation of this reaction buffer at a 1X concentration in protein phosphatase assay buffer 1 M
10. arget from the beads by adding 30 ul of 10 mM Tris HCl or 0 1X TE Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature gt Put the tube PCR plate in the magnetic stand until the solution is clear Without disturbing the bead pellet carefully transfer 25 ul of the supernatant to a fresh sterile microfuge tube Adaptor Ligation of dA Tailed DNA Mix the following components in a sterile microfuge tube dA Tailed DNA 25 pl red Quick Ligation Reaction Buffer 5X 10 p red NEBNext Adaptor 15 uM 10 ul red Quick T4 DNA Ligase 5 ul Total volume 50 ul Adaptors can be purchased separately under NEB E7335 E7350 E7500 E7600 E6609 Incubate in a thermal cycler for 15 minutes at 20 C Add 3 ul of red USER Enzyme Mix by pipetting up and down and incubate at 37 C for 15 minutes Note This step is only required for use with NEBNext Adaptors USER enzyme can be found in the NEBNext Singleplex NEB E7350 or Multiplex NEB E7335 E7500 E6609 and E7600 Oligos for Illumina A precipitate can form upon thawing of the NEBNext Q5 Hot Start HiFi PCR Master Mix To ensure optimal performance place the master mix at room temperature while performing cleanup of adaptor ligated DNA Once thawed gently mix by inverting the tube several times Cleanup of Adaptor Ligated DNA Vortex AMPure XP Beads to resuspend Add 90 ul of resuspended AMPure XP Beads to t
11. cubate at room temperature for 30 seconds and then carefully remove and discard the supernatant Repeat Step 6 once Air dry beads for 5 minutes while the tube PCR plate Is on the magnetic stand with the lid open Caution Do not overdry the beads This may result in lower recovery of DNA target Remove the tube plate from the magnet Elute the DNA target from the beads by adding 17 ul of 10 mM Tris HCl or 0 1X TE Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature Put the tube PCR plate in the magnetic stand until the solution is clear Without disturbing the bead pellet carefully transfer 15 ul of the superna tant to a clean PCR tube and proceed to enrichment PCR Enrichment of Adaptor Ligated DNA Note NEBNext Singleplex and Multiplex Oligos for Illumina NEB 7350 7335 and E7500 now have new primer concentrations 10 pM Please check oligo kit lot numbers to determine how to set up your PCR reaction Follow Section 1 8A if you are using the following oligos 10 pM primer NEBNext Singleplex Oligos for Illumina NEB E7350 lot 0071412 NEBNext Multiplex Oligos for Illumina Set 1 NEB E7335 lot 0091412 NEBNext Multiplex Oligos for Illumina Set 2 NEB E7500 lot 0071412 NEBNext Multiplex Oligos for Illumina Dual Index Primers NEB E7600 all lots Follow Section 1 8B if you are using NEBNext Multiplex Oligos for Illumina 96 Index Primers NEB E6609
12. ds NEBNext End Repair Enzyme Mix T4 Polynucleotide Kinase T4 DNA Polymerase Storage Conditions 10 mM Tris HCl 100 mM KCI 1 mM DTT 0 1 mM EDTA 50 Glycerol 0 1 Triton X 100 pH 7 4 25 C Quality Control Assays SDS PAGE Purity SDS PAGE analysis of each individual enzyme indicates gt 95 enzyme purity Endonuclease Activity Incubation of a minimum of 10 ul of this enzyme mix with 1 ug of oX174 RF DNA in assay buffer for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis Phosphatase Activity Incubation of a minimum of 10 ul of this enzyme mix in protein phosphatase assay buffer 1 M diethanolamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm Functional Activity Nucleotide Incorporation 0 2 ul of this enzyme mix incorporates 10 nmol of dNTP into acid precipitable material in a total reaction volume of 50 ul in 30 minutes at 37 C in 1X T4 DNA Polymerase Reaction Buffer with 33 uM dNTPs including H dTTP 70 pg ml denatured herring sperm DNA and 50 ug ml BSA Functional Activity Nucleotide Incorporation and Phosphorylation 5 ul of this enzyme mix repairs and phosphorylates the ends of gt 95 of 10 ug of DNA fragments containing both 3 and 5 overhangs within 30 minutes at 20 C in 1
13. e following components in a sterile microfuge tube Fragmented DNA 1 85 ul green NEBNext End Repair Reaction Buffer 10X 10 ul green NEBNext End Repair Enzyme Mix 5 ul Sterile H O variable Total volume 100 pl Incubate in a thermal cycler for 30 minutes at 20 C Cleanup Using AMPure XP Beads Beckman Coulter Inc Vortex AMPure XP Beads to resuspend Add 160 ul 1 6X of resuspended AMPure XP Beads to the ligation reac tion Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature Put the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully remove and discard the supernatant Be careful not to disturb the beads that contain the DNA targets Add 200 ul of 80 freshly prepared ethanol to the tube PCR plate while in the magnetic stand Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant Repeat Step 5 once Air dry beads for 5 minutes while the tube PCR plate is on the magnetic stand with the lid open Caution Do not overdry the beads This may result in lower recovery of DNA target Remove the tube plate from the magnet Elute the DNA target from the beads by adding 47 ul of 10 mM Tris HCl or 0 1X TE Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature Put the t
14. ersal PCR Primer 2 5 pl blue NEBNext Q5 Hot Start HiFi PCR Master Mix 25 Ul Sterile H O 5 ul Total volume 50 ul The primers are provided in NEBNext Singleplex NEB E7350 or Multiplex NEB E7335 or E7500 Oligos for Illumina For use with NEBNext Multiplex Oligos NEB 7335 or 7500 use only one Index Primer per PCR reaction 10 10 PCR cycling conditions CYCLE STEP TEMP TIME 0 0 05N Initial Denaturation 98 C 30 seconds 1 Denaturation 98 C 10 seconds 2 4 Annealing Extension 65 C 75 seconds Final Extension 65 C 5 minutes 1 Hold 4 C oo If library construction was performed with 5 yg of starting material use 2 3 cycles of amplification If starting material was 1 ug use 4 cycles of amplification However optimization of PCR cycle number may be required to avoid over amplification Proceed to Cleanup Using Ampure XP Beads in Section 1 9 Cleanup Using AMPure XP Beads Vortex AMPure XP Beads to resuspend Add 45 ul 0 9X of resuspended AMPure XP Beads to the PCR reactions 50 pl Mix well on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature Put the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully remove and discard the supernatant Be careful not to disturb the beads that contain the DNA targets Add 200 ul of freshly prepared
15. esis Phosphatase Activity Incubation of this reaction buffer at a 1X concentration in protein phosphatase assay buffer 1 M diethanolamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm Lot Controlled Quick T4 DNA Ligase E6047A 0 06 ml y E6047AA 0 3 mi Y Store at 20 C Source Purified from E coli C600 pcl857 pPLc28 lig8 2 Quality Control Assays SDS PAGE Purity SDS PAGE analysis of this enzyme indicates gt 95 enzyme purity 16 Hour Incubation 50 ul reactions containing a minimum of 2 000 units of this enzyme and 1 ug of Hindlll digested Lambda DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis 50 ul reactions containing a minimum of 2 000 units of this enzyme and 1 ug T3 DNA incubated for 16 hours at 37 C also results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis Endonuclease Activity Incubation of a minimum of 3 200 units of this enzyme with 1 ug of oX174 RF DNA in assay buffer for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis Phosphatase Activity Incubation of a minimum of 20 000 units of this enzyme in protein phosphatase assay buffer 1 M diethano
16. for 16 hours at 37 C also results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis Endonuclease Activity Incubation of this reaction buffer at a 1X concentration with 1 ug of oX174 RF DNA for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis RNase Activity Incubation of this reaction buffer at 1X concentration with 40 ng of a FAM labeled RNA transcript for 16 hours at 37 C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis Phosphatase Activity Incubation of this reaction buffer at a 1X concentration in protein phosphatase assay buffer 1 M diethanolamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm Lot Controlled 18 NEBNext Q5 Hot Start HiFi PCR Master Mix E6630A 0 3 ml Concentration 2X E6630AA 1 75 ml 2 vials provided Store at 20 C Description The NEBNext Q5 Hot Start HiFi PCR Master Mix is specifically optimized for robust high fidelity amplification of next generation sequencing NGS libraries regardless of GC content The polymerase component of the master mix Q5 High Fidelity DNA Polymerase is a novel thermostable DNA polymerase that possesses 3 55 exonuclease activity and is fused to a process
17. he ligation reaction 53 pl Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature Put the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully remove and discard the supernatant Be careful not to disturb the beads that contain the DNA targets Add 200 ul of 80 freshly prepared ethanol to the tube PCR plate while in the magnetic stand Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant 10 1 7 Repeat Step 5 once Air dry beads for 5 minutes while the tube PCR plate is on the magnetic stand with the lid open Caution Do not overdry the beads This may result in lower recovery of DNA target Remove the tube plate from the magnet Elute the DNA target by adding 105 ul of 10 mM Tris HCI or 0 1 X TE to the beads for bead based size selection Note For size selection using E Gel size select gels or standard 2 agarose gels elute the DNA target at desired volume Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature Put the tube PCR plate in the magnetic stand until the solution is clear Transfer 100 ul of supernatant or desired volume to a new tube well and proceed to bead based size selection Size Select Adaptor Ligated DNA Using AMPure XP Beads Insert Size
18. hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm Functional Activity Multiplex PCR Bead Inhibition 30 cycles of PCR amplification of 20 ng genomic DNA with and without carboxylated magnetic beads in a 50 ul reaction containing 0 5 yM 4 plex primer mix and 1X NEBNext Q5 Hot Start HiFi PCR Master Mix result in the four expected amplicons and no inhibition of amplification in the presence of the beads Lot Controlled This product is covered by one or more patents This product is licensed from Bio Rad Laboratories Inc under U S Pat Nos 6 627 424 7 541 170 7 670 808 7 666 645 and corresponding patents in other countries for use only in a standard non real time PCR in the research field only but not real time PCR or digital PCR b any in vitro diagnostics application except for applications using real time or digital PCR and c any non PCR applications in DNA sequencing isothermal amplification and the production of synthetic DNA Revision History Revision Description 4 0 N A 5 0 Include protocol for use with NEBNext Q5 Hot Start HiFi PCR Master Mix Include protocol for changes in concentration of NEBNext Singleplex and Multiplex Oligos for Illumina Changed all AMPure Bead drying times after ethanol washes to 5 minutes Changed all AMPure Bead elutions to 0 1X TE or 10 mM Tris HCI Changed ratio of AMPure Beads to 0 9X in final cleanup afte
19. ivity enhancing Sso7d domain Q5 also has an ultra low error rate gt 100 fold lower than that of Taq DNA Polymerase and 12 fold lower than that of Pyrococcus furiosus Pfu DNA Polymerase The buffer component of the master mix has been optimized for robust amplification even with GC rich amplicons and offers enhanced compatibility with a variety of beads used in typical NGS workflows These features make the NEBNext Q5 Hot Start HiFi PCR Master Mix ideal for NGS library construction This convenient 2X master mix contains dNTPs Mg and a proprietary buffer and requires only the addition of primers and DNA template for robust amplification The inclusion of the hot start aptamer allows convenient room temperature reaction set up Quality Control Assays 16 Hour Incubation A 50 pl reaction containing NEBNext Q5 Hot Start HiFi PCR Master Mix and 1 ug of Hindlll digested DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis 50 ul reactions containing NEBNext Q5 Hot Start HiFi PCR Master Mix and 1 ug of T3 DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis Phosphatase Activity Incubation of NEBNext Q5 Hot Start HiFi PCR Master Mix in protein phosphatase assay buffer 1 M diethanolamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4
20. lamine pH 9 8 and 0 5 mM MgCl containing 2 5 mM p nitrophenyl phosphate at 37 C for 4 hours yields no detectable p nitrophenylene anion as determined by spectrophotometric analysis at 405 nm RNase Activity Incubation of a minimum of 2 000 units of this enzyme with 40 ng of a FAM labeled RNA transcript for 16 hours at 37 C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis Exonuclease Activity Incubation of a minimum of 3 200 units of this enzyme with 1 pg sonicated SH DNA 10 cpm pg for 4 hours at 37 C in 50 pl reaction buffer releases lt 0 1 radioactivity Functional Activity Blunt End Ligation 50 pl reactions containing 0 5 ul Quick T4 DNA Ligase 18 ug Haelll digested oX174 and 1X T4 DNA Ligase Buffer incubated at 16 C for 7 5 min results in gt 95 of fragments ligated as determined by agarose gel electrophoresis Functional Activity Cohesive End Ligation 20 ul reactions containing 0 5 pl Quick T4 DNA Ligase 12 ug Hindlll digested lambda DNA and 1X T4 DNA Ligase Buffer incubated at 37 C overnight results in gt 95 of fragments ligated as determined by agarose gel electrophoresis Redigestion of the ligated products 50 pl reactions containing 6 ug of the ligated fragments 40 units Hindlll and 1X NEBuffer 2 incubated at 37 C for 2 hours results in no detectable undigested fragments as determined by agarose gel electrophoresis Functional Activity Adaptor Ligation 50
21. land Biolabs Japan Inc Telephone 81 0 3 5669 6191 Fax 81 0 3 5669 6192 e mail info neb japan com www nebj jp SINGAPORE New England Biolabs Pte Ltd Telephone 65 6776 0903 Fax 65 6778 9228 e mail sales ssg neb com www neb sg UNITED KINGDOM New England Biolabs UK Ltd Telephone 01462 420616 Call Free 0800 318486 Fax 01462 421057 Fax Free 0800 435682 e mail info uk neb com www neb uk com NEW ENGLAND ioLabs Version6 0 5 15
22. ll reactions containing 0 125 ul Quick T4 DNA Ligase 8 nmol 12 bp adaptor and 1X T4 DNA Ligase Buffer incubated at 16 C overnight results in no detectable unligated adaptor as determined by agarose gel electrophoresis Functional Activity Transformation After a five minute ligation of linearized dephosphory lated LITMUS 28 containing either blunt EcoRV or cohesive Hindlll ends and a mixture of compatible insert fragments transformation into chemically competent E coli DH 5 alpha cells yields a minimum of 1 x 10 recombinant transformants per pg plasmid DNA Lot Controlled References 1 Engler M J and Richardson C C 1982 In P D Boyer Ed The Enzymes Vol 5 p 3 San Diego Academic Press 16 2 Remaut E Tsao H and Fiers W 1983 Gene 22 103 113 NEBNext Quick Ligation Reaction Buffer E6048A 0 12 ml Concentration 5X E6048AA 0 6 ml Store at 20 C 1X NEBNext Quick Ligation Reaction Buffer 66 mM Tris HCl 10 mM MgCl 1 mM dithiothreitol 1 mM ATP 6 Polyethylene glycol PEG 6000 pH 7 6 25 C Quality Control Assays 16 Hour Incubation 50 ul reactions containing this reaction buffer at 1X concentration and 1 ug of Hindlll digested Lambda DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis 50 ul reactions containing this reaction buffer at 1X concentration and 1 pg T3 DNA incubated
23. r PCR reaction Added 2 minute incubation after eluting DNA from AMPure beads Changed PCR cycle number recommendations 6 0 Remove protocol for use with NEBNext High Fidelity 2X PCR Master Mix Include protocol for use with NEBNext Multiplex Oligos 96 Index Primers NEB E6609 19 USA New England Biolabs Inc 240 County Road Ipswich MA 01938 2723 Telephone 978 927 5054 Toll Free USA Orders 1 800 632 5227 Toll Free USA Tech 1 800 632 7799 Fax 978 921 1350 e mail info neb com www neb com CANADA New England Biolabs Ltd Telephone 905 665 4632 Toll Free 1 800 387 1095 Fax 905 665 4635 Fax Toll Free 1 800 563 3789 e mail info ca neb com www neb ca CHINA PEOPLE S REPUBLIC New England Biolabs Beijing Ltd Telephone 010 82378265 82378266 Fax 010 82378262 e mail info neb china com www neb china com FRANCE New England Biolabs France Free Call 0800 100 632 Free Fax 0800 100 610 e mail info fr neb com www neb online fr DNA CLONING DNA AMPLIFICATION amp PCR EPIGENETICS RNA ANALYSIS LIBRARY PREP FOR NEXT GEN SEQUENCING PROTEIN EXPRESSION amp ANALYSIS CELLULAR ANALYSIS GERMANY amp AUSTRIA New England Biolabs GmbH Telephone 49 0 69 305 23140 Free Call 0800 246 5227 Germany Free Call 00800 246 52277 Austria Fax 49 0 69 305 23149 Free Fax 0800 246 5229 Germany e mail info de neb com www neb online de JAPAN New Eng
24. s at 37 C in 1X NEBuffer 2 with 33 uM dNTPs including H dTTP 70 pg ml denatured herring sperm DNA and 50 ug ml BSA References 1 Derbyshire V et al 1988 Science 240 199 201 2 Clark J M et al 1987 J Mol Biol 198 1 123 127 NEBNext dA Tailing Reaction Buffer E6045A 0 06 ml Concentration 10X E6045AA 0 3 ml Store at 20 C 1X NEBNext dA Tailing Reaction Buffer 10 mM Tris HCl 10 mM MgCl 50 mM NaCl 1 mM DTT 0 2 mM dATP pH 7 9 25 C Quality Control Assays 16 Hour Incubation 50 ul reactions containing this reaction buffer at 1X concentration and 1 Wg of Hindlll digested Lambda DNA incubated for 16 hours at 37 C results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis 50 ul reactions containing this reaction buffer at 1X concentration and 1 pg T3 DNA incubated for 16 hours at 37 C also results in no detectable non specific nuclease degradation as determined by agarose gel electrophoresis Endonuclease Activity Incubation of this reaction buffer at a 1X concentration with 1 ug of oX174 RF DNA for 4 hours at 37 C in 50 ul reactions results in less than 10 conversion to RF II as determined by agarose gel electrophoresis RNase Activity Incubation of this reaction buffer at 1X concentration with 40 ng of a FAM labeled RNA transcript for 16 hours at 37 C results in no detectable RNase activity as determined by polyacrylamide gel electrophor
25. ube PCR plate in the magnetic stand until the solution is Cclear Without disturbing the bead pellet carefully transfer 42 ul of the supernatant to a fresh sterile microfuge tube dA Tailing of End Repaired DNA Mix the following components in a sterile microfuge tube End Repaired Blunt DNA 42 ul O yellow NEBNext dA Tailing Reaction Buffer 10X 5 pl O yellow Klenow Fragment 3 5 exo 3 ul Total volume 50 ul Incubate in a thermal cycler for 30 minutes at 37 C Cleanup Using AMPure XP Beads Vortex AMPure XP Beads to resuspend Add 90 ul 1 8X of resuspended AMPure XP Beads to the ligation reac tion Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature Put the tube PCR plate on an appropriate magnetic stand to separate beads from supernatant After the solution is clear about 5 minutes carefully remove and discard the supernatant Be careful not to disturb the beads that contain the DNA targets Add 200 ul of 80 freshly prepared ethanol to the tube PCR plate while in the magnetic stand Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant Repeat Step 5 once Air dry beads for 5 minutes while the tube PCR plate is on the magnetic stand with the lid open Caution Do not overdry the beads This may result in lower recovery of DNA target Remove the tube plate from the magnet Elute the DNA t
26. y Prep Master Mix Set for Illumina contains enzymes and buffers in convenient master mix formulations that are ideally suited for sample preparation for next generation sequencing and for preparation of expression libraries Each of these components must pass rigorous quality control standards and are lot controlled both individually and as a set of reagents Lot Control The lots provided in the NEBNext DNA Library Prep Master Mix Set for Illumina are managed separately and are qualified by additional functional validation Individual reagents undergo standard enzyme activity and quality control assays and also meet stringent criteria in the additional quality controls listed on each individual component page Functional Validation Each set of reagents is functionally validated together through construction and sequencing of a genomic DNA library on an Illumina Sequencer Illumina Inc For larger volume requirements customized and bulk packaging is available by purchasing through the OEM Bulks department at NEB Please contact OEM neb com for further information Protocols Symbols Ad This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable like the amount of input DNA Colored bullets indicate the cap color of the reagent to be added to a reaction Starting Material 1 5 ug of Fragmented DNA 1 1 i End Repair of Fragmented DNA Mix th
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