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Description:
The DNA Sample Prep Reagent Set 1 contains enzymes and buffers 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 DNA Sample Prep Reagent Set 1 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 section.

Each set of reagents is functionally validated together through construction and sequencing of a genomic DNA library on an Illumina Genome Analyzer II, and by construction of an expression library.

The Reagent Set Includes:

The volumes provided are sufficient for preparation of up to 10 reactions (NEB #E6000S) and 50 reactions (NEB #E6000L). (All reagents should be stored at -20°C).

• Phosphorylation Buffer (10X)
• Deoxynucleotide Solution Mix (10 mM each dNTP)
• T4 DNA Polymerase
• DNA Polymerase I, Large (Klenow) Fragment
• T4 Polynucleotide Kinase
• Deoxyadenosine 5´- Triphosphate (dATP) (1.0 mM)
• Klenow Fragment (3´→ 5´ exo^-)
• NEBuffer 2 for Klenow exo^- (10X)
• Quick T4 DNA Ligase
• Quick Ligation Reaction Buffer (2X)
• Phusion® High-Fidelity PCR Master Mix (2X) (manufactured by Finnzymes Oy)

Notes

Usage notes:

1. Refer to your specific sample preparation protocol to determine conditions for use.

FAQs

1. Do you provide a protocol for using the NEBNext SPRS 1 reagents?
2. What type of sample prep/library construction can I use the NEBNext SPRS 1 reagents for?
3. What type and how much starting material do I need to use when preparing libraries using the NEBNext DNA SPRS 1?
4. Can I use the NEBNext SPRS 1 reagents for preparing RNA? For miRNA work?
5. Can I use these reagents for preparing libraries for sequencing on the ABI SOLiD instrument?
6. Can NEB provide any of the reagents for the cluster generation step or the actual sequencing run?
7. Can I use the NEBNext SPRS 1 reagents for preparing libraries for sequencing on the Roche 454 GSFLX?
8. Are these reagents available in a customized format, larger format or 96 well format?
9. Will use of these reagents void the warranty on my Illumina instrument?
10. Do I need to dA-tail my insert for cloning an expression library?
11. What are the differences between expression library preparation vs. sample preparation suitable for the Illumina platform?

Protocols

Protocols for NEBNext™ DNA Sample Prep Reagent Set 1

Quality Control for Current Lot

Quality control values for a specific lot can be found on the datacard which accompanies each product.

QC for Phosphorylation Buffer:

16-Hour Incubation: 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg 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 μg of φX174 RF I DNA for 4 hours at 37°C in 50 μl 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 (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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.

QC for Deoxynucleotide Solution Mix:

16-Hour Incubation: 50 μl reactions containing a minimum of 2 mM dNTPs and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing a minimum of 2 mM dNTPs and 1 μg T3 DNA incubated for 16 hours at 37°C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.

RNase Activity: Incubation of 1 mM dNTPs 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 a minimum of 5 mM dNTPs in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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.

HPLC: dNTP purity is determined by HPLC to be >99%.

Functional Activity (PCR): The dNTPs are tested in 25 cycles of PCR amplification generating 0.5 kb, 2 kb, and 5kb amplicons from lambda DNA.

QC for T4 DNA Polymerase:

SDS-PAGE Purity: SDS-PAGE analysis of this enzyme indicates >95% enzyme purity.

Endonuclease Activity: Incubation of a minimum of 50 units of this enzyme with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl reactions results in less than 10% conversion to RF II as determined by agarose gel electrophoresis.

Phosphatase Activity: Incubation of a minimum of 30 units of this enzyme in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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): One unit of this enzyme incorporates 10 nmol of dNTP into acid-precipitable material in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X T4 DNA Polymerase Reaction Buffer with 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA and 50 µg/ml BSA.

QC for DNA Polymerase I, Large (Klenow) Fragment:

SDS-PAGE Purity: SDS-PAGE analysis of this enzyme indicates >95% enzyme purity.

16-Hour Incubation: 50 μl reactions containing a minimum of 5 units of this enzyme and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing a minimum of 5 units of this enzyme and 1 μg 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 50 units of this enzyme with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl 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 (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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 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.

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 μl in 30 minutes at 37°C in 1X NEBuffer 2 with 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA and 50 µ g/ml BSA.

QC for dATP Solution:

Phosphatase Activity: Incubation of a minimum of 1 mM dATP in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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.

16-Hour Incubation: 50 μl reactions containing a minimum of 0.2 mM dATP and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing 0.2 mM dATP and 1 μg T3 DNA incubated for 16 hours also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.

RNase Activity: Incubation of a minimum of 0.1 mM dATP 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.

HPLC: dATP purity is determined by HPLC to be >99%.

Functional Activity (PCR): This dATP in a pool of dNTPs is tested in 25 cycles of PCR amplification generating 0.5 kb, 2 kb, and 5kb amplicons from lambda DNA.

QC for Klenow Fragment (3´→ 5´ exo-):

SDS-PAGE Purity: SDS-PAGE analysis of this enzyme indicates >95% enzyme purity.

16-Hour Incubation: 50 μl reactions containing a minimum of 5 units of this enzyme and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing a minimum of 5 units of this enzyme and 1 μg 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 50 units of this enzyme with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl 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 (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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 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 μg sonicated [3H]DNA (105 cpm/μg) for 4 hours at 37°C in 50 μL reaction buffer releases < 0.1% radioactivity.

3´→5´ Exonuclease Activity: Incubation of a minimum of 50 units of enzyme in 20 μl 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 μl in 30 minutes at 37°C in 1X NEBuffer 2 with 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA and 50 µg/ml BSA.

QC for NEBuffer 2 for Klenow exo-:

16-Hour Incubation: 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg 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 μg of φX174 RF I DNA for 4 hours at 37°C in 50 μl 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 (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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.

QC for Quick T4 DNA Ligase:

SDS-PAGE Purity: SDS-PAGE analysis of this enzyme indicates >95% enzyme purity.
16-Hour Incubation: 50 μl reactions containing a minimum of 2,000 units of this enzyme and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing a minimum of 2,000 units of this enzyme and 1 μg 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 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl 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 (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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 μg sonicated [3H]DNA (105 cpm/μg) for 4 hours at 37°C in 50 μL reaction buffer releases < 0.1% radioactivity.

Functional Activity (Blunt End Ligation): 50 μl reactions containing a 0.5 uL Quick T4 DNA Ligase, 18 μg HaeIII digested φX174 and 1X T4 DNA Ligase Buffer incubated at 16°C for 7.5 min results in >95% of fragments ligated as determined by agarose gel electrophoresis.

Functional Activity (Cohesive End Ligation): 20 μl reactions containing 0.5 uL Quick T4 DNA Ligase, 12 μg HindIII digested lambda DNA and 1X T4 DNA Ligase Buffer incubated at 37°C overnight results in >95% of fragments ligated as determined by agarose gel electrophoresis. Redigestion of the ligated products, 50 μl reactions containing 6 μg of the ligated fragments, 40 units HindIII, 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 (Adapter Ligation): 50 μl reactions containing 0.125 uL Quick T4 DNA Ligase, 8 nmol 12 bp adapter, and 1X T4 DNA Ligase Buffer incubated at 16°C overnight results in no detectable unligated adapter as determined by agarose gel electrophoresis.

Functional Activity (Transformation): After a five-minute ligation of linearized, dephosphorylated LITMUS 28 (containing either blunt [EcoRV] or cohesive [HindIII] ends) and a mixture of compatible insert fragments, transformation into chemically competent E. coli DH-5 alpha cells yields a minimum of 1 x 106 recombinant transformants per ug plasmid DNA.

QC for Quick Ligation Reaction Buffer:

Endonuclease Activity: Incubation of this reaction buffer at a 1X concentration with 1 μg of φX174 RF I DNA for 4 hours at 37°C in 50 μl 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.

QC for T4 Polynucleotide Kinase:

SDS-PAGE Purity: SDS-PAGE analysis of this enzyme indicates >95% enzyme purity.

16-Hour Incubation: 50 μl reactions containing a minimum of 10 units of this enzyme and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing a minimum of 10 units of this enzyme and 1 μg 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 200 units of this enzyme with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl reactions results in less than 10% conversion to RF II as determined by agarose gel electrophoresis.

Phosphatase Activity: Incubation of a minimum of 100 units of this enzyme in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) 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 100 units of this enzyme with 2 μg MS2 phage RNA for 1 hour at 37°C in 50 μl 1X T4 Polynucleotide Kinase Reaction Buffer followed by agarose gel electrophoresis shows no degradation. Incubation of 10 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 300 units of enzyme with 1 μg sonicated [3H]DNA
(105 cpm/μg) for 4 hours at 37°C in 50 μL reaction buffer released < 0.1% radioactivity.

Functional Activity (Labeling): 32P end labeling of 5´-hydroxyl terminated d(T)8 with a minimum of 50 units of this enzyme for 30 minutes at 37°C in 50 µl 1X T4 Polynucleotide Kinase Buffer followed by 20% acrylamide gel electrophoresis reveals that less than 1% of the product has been degraded by exonuclease or phosphatase activities.

QC for Phusion® High-Fidelity PCR Master Mix:

16-Hour Incubation: 50 μl reactions containing 1X Phusion Master Mix and 1 μg of HaeIII digested φX174 RF I DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing 1X Phusion Master Mix and 1 μg T3 DNA incubated for 16 hours at 37°C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.

Phosphatase Activity: Incubation of 1X Phusion Master Mix in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) 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 (PCR): The 1X Phusion High-Fidelity PCR Master Mix is tested in 35 cycles of PCR amplification with a series of primers generating 196-723 bp amplicons from human genomic DNA.

Quality Control for Current Lot

NEBNext™ dA-Tailing Module
NEBNext™ DNA Sample Prep Master Mix Set 1
NEBNext™ dsDNA Fragmentase™
NEBNext™ End Repair Module
NEBNext™ Quick Ligation Module