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Stratagene superscript iii reverse transcriptase
OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated <t>RNA</t> probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed <t>three</t> and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.
Superscript Iii Reverse Transcriptase, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination"

Article Title: AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1335

OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated RNA probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed three and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.
Figure Legend Snippet: OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated RNA probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed three and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.

Techniques Used: Isolation, Transfection, Selection, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Derivative Assay, Construct, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

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Amplification:

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Isolation:

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Northern Blot:

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Purification:

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Real-time Polymerase Chain Reaction:

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Polymerase Chain Reaction:

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Article Title: AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination
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Sequencing:

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    Stratagene superscript iii reverse transcriptase
    OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated <t>RNA</t> probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed <t>three</t> and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.
    Superscript Iii Reverse Transcriptase, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Stratagene
    Average 86 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2020-04
    86/100 stars
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    OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated RNA probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed three and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.

    Journal: Nucleic Acids Research

    Article Title: AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

    doi: 10.1093/nar/gks1335

    Figure Lengend Snippet: OligoA mutations change CoTC cleavage distribution. ( A ) The hscRACE procedure. Terminator element (black box), exon 3 (grey box) and cleavage sites (lightning bolts) are shown. Biotinylated RNA probe, β3 (tailed box), was hybridized to nuclear RNA isolated from HeLa cells transfected with pβT+ or pβTa- > c and then isolated by magnetic selection. Selected RNA is subjected to oligonucleotide (dotted line)-directed RNAse H digestion releasing RNA from beads. This is ligated and reversed transcribed across the ligated junction. cDNA is then amplified by PCR using gene-specific primers. The PCR products are analysed on a gel, cloned and sequenced. ( B ) Terminator DNA sequence showing the positions of CoTC-derived RNA 3′-ends in pβT+ (black filled arrows) and pβTa- > c (empty arrows). Positions of AA to CC mutations in pβTa- > c are underlined. The numbers above each arrow indicate the number of clones identified for each site. The hscRACE experiment was performed three and seven times for pβT+ and pβTa- > c, respectively, in order to obtain fifty positive colonies for each construct. ( C ) Effect of siRNA-mediated knock down of CPSF73 and CstF64. pβT+ΔpA has a mutated PAS (AATAAA- > GAATTC), known to inactivate β-globin mRNA polyadenylation (Dye and Proudfoot, 1999). This was transfected into CPSF73 and CstF64 siRNA-treated and mock-treated cells. Relative RNA continuity (based on RT/PCR analysis as in Figure 2 A) was assessed and is presented graphically. Error bars denote standard deviation.

    Article Snippet: RNA analysis One microgram of RNA was reverse transcribed with Superscript III Reverse Transcriptase (Stratagene) followed by quantitative PCR with a QuantiTect SYBR green kit (Qiagen) on a Corbett Rotor Gene 3000 machine using primers listed ( Supplementary Table S1 ).

    Techniques: Isolation, Transfection, Selection, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Derivative Assay, Construct, Reverse Transcription Polymerase Chain Reaction, Standard Deviation