Buffer e

Manufactured by Promega

Sourced in United States

Buffer E is a laboratory reagent used to maintain the pH and ionic conditions of biological samples during various experimental procedures. It is a concentrated solution that can be diluted as needed to create the desired buffer system for the specific application.

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Lab products found in correlation

Bamhi, by Promega (3 mentions) Fragmentation reagent, by Thermo Fisher Scientific (3 mentions) Circligase 2, by Illumina (3 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions)

4 protocols using buffer e

RNA-seq library preparation protocol

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10 μg total RNA extracted with Trizol (Ambion) was fragmented with fragmentation reagent (Ambion) at 70°C for 10 min followed by precipitation with ethanol. Reverse transcription was performed with PASSEQ7-2 RT oligo:
and Superscript III (Invitrogen). cDNA was recovered by ethanol precipitation and 120–200 nucleotides of cDNA was gel-purified from 8% urea–PAGE. Recovered cDNA was circularized with Circligase™ II (Epicentre) at 60°C overnight. Buffer E (Promega) was added to the cDNA and heated at 95°C for 2 min, and then cool to 37°C slowly. Circularized cDNA was linearized by BamH I (Promega). cDNA was collected by centrifugation after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index (Illumina). Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).

Chan D., Feng C., England W.E., Wyman D., Flynn R.A., Wang X., Shi Y., Mortazavi A, & Spitale R.C. (2021). Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing. Nucleic Acids Research, 49(20), 11868-11882.

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RNA-seq Library Preparation Protocol

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Total RNA was extracted with Trizol as per manual (Life technologies), 10 μg total RNA was fragmented with fragmentation reagent (Ambion) at 70 °C for 10 minutes followed by precipitation with ethanol. After centrifugation, RNA was dissolved and Reverse transcription was performed with PASSEQ7-2 RT oligo:[phos]NNNNAGATCGGAAGAGCGTCGTGTTCGGATCCATTAGGATCCGAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTT[V-Q] and Superscript III. cDNA was recovered by ethanol precipitation and centrifugation. 120–200 nucleotides of cDNA was gel-purified and eluted from 8% Urea-PAGE. Recovered cDNA was circularized with Circligase^™ II (Epicentre) at 60 °C overnight. Buffer E (Promega) was added in cDNA and heated at 95 °C for 2 minutes, and then cool to 37 °C slowly. Circularized cDNA was linearized by BamH I (Promega). cDNA was collected by centrifugation after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index. Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).

Zhu Y., Wang X., Forouzmand E., Jeong J., Qiao F., Sowd G.A., Engelman A.N., Xie X., Hertel K.J, & Shi Y. (2017). Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation. Molecular cell, 69(1), 62-74.e4.

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Poly(A) Site Mapping Protocol

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Poly(A) site mapping was carried out as previously described (Spies et al., 2013 (link)). Total RNA was extracted with Trizol as per the manufacturer’s recommendations (Life Technologies). Then, 10 μg of total RNA was fragmented with fragmentation reagent (Ambion) at 70°C for 10 minutes followed by precipitation with ethanol. After centrifugation, RNA was dissolved and reverse transcription was performed using Superscript III (Thermo Fisher Scientific) using a custom primer, PASSEQ7-2 RT oligo: [phos]NNNNAGATCGGAAGAGCGTCGTGTTCGGATCCATTAGGATCCGAGAC GTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTT[V-Q]. cDNA was recovered by ethanol precipitation and centrifugation. cDNA ranging from 120–200 basepairs was gel-purified and eluted from 8% Urea-PAGE. Recovered cDNA was circularized with Circligase^™ II (Epicentre) at 60 °C overnight. Buffer E (Promega) was added to cDNA and heated at 95°C for 2 minutes, and then slowly cooled to 37°C. Circularized cDNA was linear ized by adding BamHI (Promega). cDNA was centrifuged after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index. Around 200 basepair of PCR product was gel-purified and submitted for sequencing (single end, 100 nucleotides).

Brumbaugh J., Di Stefano B., Wang X., Borkent M., Forouzmand E., Clowers K.J., Ji F., Schwarz B.A., Kalocsay M., Elledge S.J., Chen Y., Sadreyev R.I., Gygi S.P., Hu G., Shi Y, & Hochedlinger K. (2017). Nudt21 controls cell fate by connecting alternative polyadenylation to chromatin signaling. Cell, 172(1-2), 106-120.e21.

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HTLV-1/2 Identification via RFLP

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After the identification of cases with positive PCR results, using the 1% agarose gel, enzymatic digestion (RFLP—polymorphisms of the length of restriction fragments) of the amplified product was performed to identify the type of HTLV-1/2 present in the samples.
The RFLP reaction of this pX gene product (159 bp) was performed by mixing 6.0 μL of the amplified product, 7 μL of H20, 1.5 μL of buffer E (Promega, Madison WI, USA), and 0.5 μL of the restriction enzyme TaqI (10 U/μL, Promega, Madison WI, USA), with subsequent incubation at 65°C for 2 h.
The presence of the restriction site (T/CGA) generates three fragments (21, 53, and 85 bp) in HTLV-2 and two fragments (21 and 138 bp) in HTLV-1.
The products of enzymatic digestion were visualized in agarose gel of 3% to 100 V for 45 min in 1 × TAE buffer (TAE 50 × stock-TrisBase 1.6 M, Na Acetate 0.8 M, and EDTA-Na240 Mm/1,000 ml of deionized water), using transilluminator in light with an ultra-violet light source (Tuke et al., 1992 (link)).

Ribeiro J.F., Nobre A.F., Covre L.C., de Almeida Viana M.D., Silva I.C., dos Santos L.M., Ishikawa E.A., da Costa C.A, & de Sousa M.S. (2022). Hematological changes in human lymphotropic-T virus type 1 carriers. Frontiers in Microbiology, 13, 1003047.

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