Neb buffer 4

Manufactured by New England Biolabs

Sourced in United States

NEB Buffer 4 is a DNA digestion buffer solution provided by New England Biolabs. It is designed to be used with specific restriction enzymes to facilitate DNA cleavage and manipulation.

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Lab products found in correlation

9 protocols using neb buffer 4

Genotyping of Sirpa and Mertk SNPs

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For Sirpa, the rs108285434 SNP was analysed as previously described [18 (link)]. An amplicon polymorphic between B6 and 129 was generated by PCR from mouse gDNA using the following primers: forward 5’-CCGTTCTGAACTGCACTTTG-3’ and reverse 5’-GGGGTGACATTACTGATACGG-3’ with the following cycling conditions: 2 minutes at 94°C and then 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, 40 seconds at 72°C and then 7 minutes at 72°C. Amplicons were directly digested with AvaI enzyme in the presence of NEB buffer 4 (both from New England Biolabs) for 2 h at 37°C and digestion products were visualized by agarose gel electrophoresis.
For Mertk, the rs27446500 SNP was analysed. An amplicon polymorphic between B6 and 129 was generated by PCR from mouse gDNA using the following primers: forward 5’-TTGGGTTTCATCCCATCCCC-3’ and reverse 5’-GGCACACCTAATCCCCAACT-3’ with the following cycling conditions: 2 minutes at 94°C and then 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, 40 seconds at 72°C and then 7 minutes at 72°C. Amplicons were directly digested with Hyp188I enzyme in the presence of NEB buffer 4 (both from New England Biolabs) for 2 h at 37°C and digestion products were visualized by agarose gel electrophoresis.

Nuvolone M., Paolucci M., Sorce S., Kana V., Moos R., Matozaki T, & Aguzzi A. (2017). Prion pathogenesis is unaltered in the absence of SIRPα-mediated "don't-eat-me" signaling. PLoS ONE, 12(5), e0177876.

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Expression Vector Construction Protocol

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Comprehensive design and construction details for all expression vectors are provided in Table 1. The assembly of some plasmids required annealing of complementary oligonucleotides: 50 pmol of each oligonucleotide was mixed in 50 μl ddH₂O-diluted 1x NEB Buffer 4 (New England Biolabs, Ipswich, MA, USA), heated for 10 min at 95°C, cooled down over 4 h to 22°C and incubated at 22°C for another 2 h prior to cloning into the corresponding vector backbone. All relevant genetic components have been confirmed by sequencing (Microsynth, Balgach, Switzerland).

Wang H., Ye H., Xie M., Daoud El-Baba M, & Fussenegger M. (2015). Cosmetics-triggered percutaneous remote control of transgene expression in mice. Nucleic Acids Research, 43(14), e91.

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Plasmid Construction and Verification

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Comprehensive design and construction details for all expression vectors are provided in Table 1. The assembly of some plasmids required annealing complementary oligonucleotides. For optimal annealing, 50 pmol of each oligonucleotide was mixed in 50 μl ddH₂O-diluted 1x NEB Buffer 4 (New England Biolabs, Ipswich, MA, USA), heated for 10 min at 95°C, cooled down over 4 h to 22°C and incubated at 22°C for another 2 h prior to cloning into the corresponding vector backbone. All relevant genetic components have been confirmed by sequencing (Microsynth, Balgach, Switzerland).

Xie M., Ye H., Hamri G.C, & Fussenegger M. (2014). Antagonistic control of a dual-input mammalian gene switch by food additives. Nucleic Acids Research, 42(14), e116.

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Biotinylated cDNA Hybridization Assay

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The biotinylated cDNA (approx. 100 pmol or 3.5 µg) is hybridized with 2.5-foldexcess Nb.BtsI forward restriction oligo (P6) and 1.5-fold BspQI reverse restriction oligo (P7). The hybridization is done in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (1x NEB buffer 4; New England biolabs) solution supplemented with 1x bovine albumin serum. The solution is incubated at 80°C for 2 min to melt DNA strands, followed by slow cooling (−0.1°C/s) to 37°C (50 ul)

Murgha Y.E., Rouillard J.M, & Gulari E. (2014). Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries. PLoS ONE, 9(4), e94752.

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Restriction Digest and Adapter Ligation Protocol

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Restriction digestion was performed using 1.25 µl NEB Buffer 4 (New England Biolabs, Foster City, CA), 0.125 µl bovine serum albumin (New England Biolabs), 0.0625 µl EcoRI, 0.0625 µl MseI (New England Biolabs), 3.94 µl Nanopure^® water and 7 µl of ∼20 ng/µl DNA template for a total volume of 12.5 µl. The restriction digestion was incubated on a GeneAmp 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA) at 37 °C for 2.5 h. A ligation mixture (5 µl) consisting of 0.5 µl EcoRI and MseI prepared adapters, (Integrated DNA Technologies, Coralville, IA, USA), 0.5 µl T4 DNA ligase, 0.15 µl 10× T4 DNA ligase buffer (New England Biolabs), and 3.35 µl Nanopure^® water was dispensed into the tubes containing the digestion product and incubated at 25 °C for 8 h. The ligation product was then diluted using 135 µl of 1× TE buffer. A Nanodrop^® spectrophotometer (Thermo Fisher Scientific, Walltham, MA, USA) was used to determine the quantity and quality of DNA in ng/µl from each tube.

Ullah M.I., Mustafa F., Kneeland K.M., Brust M.L., Hoback W.W., Kamble S.T, & Foster J.E. (2014). Forms of Melanoplus bowditchi (Orthoptera: Acrididae) collected from different host plants are indistinguishable genetically and in aedeagal morphology. PeerJ, 2, e418.

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Methylation Profiling Using AFSM Method

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We chose a combination of two restriction enzyme pairs, EcoRI-MspI and EcoRI-HpaII. The isoschizomers HpaII and MspI were used as frequent cutters, while EcoRI was used as a rare cutter. Methylation-susceptible, anonymous 5′-CCGG sequences and their methylation statuses can be assessed by the AFSM method. To detect the AFSM sites, two digestion reactions were set up at the same time. In the first reaction, genomic DNA (200 ng) was digested in a 20 μl reaction volume of NEB Buffer 4 with 10 U of EcoRI (New England BioLabs Inc., Ipswich, MA, R0101) and 10 U of MspI (New England BioLabs Inc., Ipswich, MA, R0106). The second digestion reaction was carried in exactly the same manner except that HpaII and NEB Buffer 1 were used in place of MspI and NEB Buffer 4. The two sets of DNA samples were digested for 8 h at 37°C. Then, incubation was carried out at 65°C for 30 min to inactivate the enzymes.

Xia Z., Zou M., Zhang S., Feng B, & Wang W. (2014). AFSM sequencing approach: a simple and rapid method for genome-wide SNP and methylation site discovery and genetic mapping. Scientific Reports, 4, 7300.

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Plasmid DNA Isolation and Purification

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Yeast colonies were picked into 1.5 ml of the appropriate SD medium in a 2-ml deep-well block and incubated with shaking at 30°C and 1000 rpm until grown to saturation, typically 2 days. Cell pellets were harvested through centrifugation at 2800g for 20 min, and the supernatants were discarded. Plasmid DNA was isolated using a modified version of the QIAprep Turbo miniprep kit (27173/27191/27193, Qiagen). Zymolase (1000 U; Zymo Research E1004/5) and 80 mg of RNAse A (19101, Qiagen) were added to 400 ml of P1 buffer. Each pellet in the deep-well block was resuspended in 250 μl of this modified P1 and incubated with shaking for 2 to 3 hours. The remainder of the plasmid preparation was undertaken according to the manufacturer’s instructions with final elution in 100 μl of water.
Prepared plasmids were treated with exonuclease to remove any contaminating linear DNA. A volume of 22.5 μl of each prepared plasmid was combined with 1.5 μl of Exonuclease V (10 U/μl; NEB, M0345L), 3.0 μl of NEB Buffer 4 (NEB, B7004S), and 3.0 μl of ATP (10 mM; NEB, P0756S) followed by incubation at 37°C for 1 hour. Exonuclease reactions were quenched by the addition of 1 μl of EDTA (0.33 M) and incubation for 30 min at 70°C. Finally, reactions were purified using Sera-mag magnetic particles (1.5 mg/ml, bead/sample ratio = 1:1) with final elution in 25 μl of tris-Cl (10 mM).

Harvey C.J., Tang M., Schlecht U., Horecka J., Fischer C.R., Lin H.C., Li J., Naughton B., Cherry J., Miranda M., Li Y.F., Chu A.M., Hennessy J.R., Vandova G.A., Inglis D., Aiyar R.S., Steinmetz L.M., Davis R.W., Medema M.H., Sattely E., Khosla C., St. Onge R.P., Tang Y, & Hillenmeyer M.E. (2018). HEx: A heterologous expression platform for the discovery of fungal natural products. Science Advances, 4(4), eaar5459.

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USER Cloning and Fusion Protocol

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USER cloning and USER fusion was performed as previously described [9] (link), [11] (link) with minor modifications: Vector backbones holding a PacI/Nt.BbvCI USER cloning compatible cassette were digested as previously described [13] (link). 1 µl of USER enzyme mix, 0.5 µl NEB Buffer 4, 0.5 µl BSA x10 (all purchased from New England Biolabs), and 0.1 pmol digested vector backbone were mixed in a 0.2 ml PCR tube. If vector backbone was amplified by PCR, 1 µl purified DNA element was added. Finally, 7 µl of purified DNA elements were mixed to a total reaction volume of 10 µl. If more than one DNA element was to be inserted, all elements were added in equal volumes. The reaction mixture was incubated for 40 min at 37°C, followed by 30 min at 25°C. Subsequently the 10 µl reaction mixture was used directly to transform chemically competent E. coli DH5α cells. Transformants were selected in Luria Broth (LB) medium supplemented with 100 µg/ml ampicillin. Three transformants were picked randomly for each construct and validated by DNA sequencing (Star SEQ, Mainz, Germany). For each construct, a validated expression vector was purified by Plasmid Plus Maxi kit (Qiagen, Hilden, Germany) following the manufacturer's instruction and dilution to a concentration of 1 µg/µl DNA in MilliQ water.

Lund A.M., Kildegaard H.F., Petersen M.B., Rank J., Hansen B.G., Andersen M.R, & Mortensen U.H. (2014). A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering. PLoS ONE, 9(5), e96693.

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Microarray Study with MSRE Digestion

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For the microarray study, 3 µg of genomic NA was digested at 37 degrees Celsius for 16 hrs with an in the methyl-sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 µl reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. Samples were ethanol precipitated, washed in 70% ethanol and resuspended in reduced EDTA TE (5 mM Tris, 0.1 mM EDTA) at 50 ng/µl. All samples underwent the regular Affymetrix SNP 6.0 hybridization procedure in duplicate; samples were digested with the Nsp I and StyI restriction enzymes, fragments of 100–1200 bp (containing the polymorphic sites to be assessed) PCR amplified, and the resulting amplicons labeled and hybridized to the array.

Hutchinson J.N., Raj T., Fagerness J., Stahl E., Viloria F.T., Gimelbrant A., Seddon J., Daly M., Chess A, & Plenge R. (2014). Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease. PLoS ONE, 9(6), e98464.

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