3 end blocked dna linker Search Results


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  • 99
    New England Biolabs λ phage dna
    λ Phage Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/λ phage dna/product/New England Biolabs
    Average 99 stars, based on 125 article reviews
    Price from $9.99 to $1999.99
    λ phage dna - by Bioz Stars, 2020-07
    99/100 stars
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    99
    Thermo Fisher ut4 dna ligase pmolrna
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Ut4 Dna Ligase Pmolrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1136 article reviews
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    ut4 dna ligase pmolrna - by Bioz Stars, 2020-07
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    93
    Integrated DNA Technologies 3 end blocked linker 1
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    3 End Blocked Linker 1, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3 end blocked linker 1/product/Integrated DNA Technologies
    Average 93 stars, based on 6 article reviews
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    3 end blocked linker 1 - by Bioz Stars, 2020-07
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    99
    Millipore dna fragment
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Dna Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna fragment/product/Millipore
    Average 99 stars, based on 4587 article reviews
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    dna fragment - by Bioz Stars, 2020-07
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    96
    Millipore salmon sperm dna
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Salmon Sperm Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 4224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    salmon sperm dna - by Bioz Stars, 2020-07
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    99
    Bio-Rad dna engine
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Dna Engine, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna engine/product/Bio-Rad
    Average 99 stars, based on 426 article reviews
    Price from $9.99 to $1999.99
    dna engine - by Bioz Stars, 2020-07
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    99
    Thermo Fisher lambda dna
    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by <t>T4</t> DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Lambda Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lambda dna/product/Thermo Fisher
    Average 99 stars, based on 201 article reviews
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    99
    TaKaRa t4 dna ligase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 dna ligase - by Bioz Stars, 2020-07
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    94
    New England Biolabs therminator dna polymerase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Therminator Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 102 article reviews
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    therminator dna polymerase - by Bioz Stars, 2020-07
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    99
    New England Biolabs t4 dna polymerase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 9070 article reviews
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    t4 dna polymerase - by Bioz Stars, 2020-07
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    96
    Thermo Fisher t4 dna ligase buffer
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
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    t4 dna ligase buffer - by Bioz Stars, 2020-07
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    99
    Thermo Fisher taq dna polymerase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 44901 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2020-07
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    99
    New England Biolabs phusion dna polymerase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
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    New England Biolabs dna polymerase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra ii fs dna library prep kit for illumina
    PCR primers used in the amplification reaction, and their cognate substrates. We amplify both LTR/host junctions and the primers for both ends of the provirus are shown. Panel a . At the top is a drawing of the T-linker, and the first and second linker primers. The blocking group (NH3, C6) on the 3′ end of the short strand of the T-linker is shown, and the 5′ end of short strand is phosphorylated (P), to allow it to be ligated to the host <t>DNA.</t> The second linker primer is used to add the sequences needed for <t>Illumina</t> sequencing to one end of the amplified fragment. The first and second LTR primers used to amplify the 5′ LTR are shown in the drawing, with the linker primers used to amplify the host-virus DNA junction. The sequences needed for Illumina sequencing to the other end of the amplified fragments are added as part of the second LTR primer. Panel b shows the amplification of the 3′ LTR and the appended host sequences, together with the first and second LTR primers (see also Fig. 2 ). Panel c At the bottom of the drawing, are the corresponding primers used to amplify the 5′ LTR
    Nebnext Ultra Ii Fs Dna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion hot start ii dna polymerase
    PCR primers used in the amplification reaction, and their cognate substrates. We amplify both LTR/host junctions and the primers for both ends of the provirus are shown. Panel a . At the top is a drawing of the T-linker, and the first and second linker primers. The blocking group (NH3, C6) on the 3′ end of the short strand of the T-linker is shown, and the 5′ end of short strand is phosphorylated (P), to allow it to be ligated to the host <t>DNA.</t> The second linker primer is used to add the sequences needed for <t>Illumina</t> sequencing to one end of the amplified fragment. The first and second LTR primers used to amplify the 5′ LTR are shown in the drawing, with the linker primers used to amplify the host-virus DNA junction. The sequences needed for Illumina sequencing to the other end of the amplified fragments are added as part of the second LTR primer. Panel b shows the amplification of the 3′ LTR and the appended host sequences, together with the first and second LTR primers (see also Fig. 2 ). Panel c At the bottom of the drawing, are the corresponding primers used to amplify the 5′ LTR
    Phusion Hot Start Ii Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Integrated DNA Technologies 3 end rna linker
    Properties of the cleavage activity. <t>RNA</t> substrates and cleavage sites are indicated at the left-hand side of gels. A, Cleavage of STS and its <t>3′-end</t> extension variants. The total lengths are the STS plus the indicated numbers of nts on the top
    3 End Rna Linker, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrated DNA Technologies dideoxy c base
    Properties of the cleavage activity. <t>RNA</t> substrates and cleavage sites are indicated at the left-hand side of gels. A, Cleavage of STS and its <t>3′-end</t> extension variants. The total lengths are the STS plus the indicated numbers of nts on the top
    Dideoxy C Base, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by T4 DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.

    Journal: Nature methods

    Article Title: Highly multiplexed simultaneous detection of RNAs and proteins in single cells

    doi: 10.1038/nmeth.3742

    Figure Lengend Snippet: PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by T4 DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.

    Article Snippet: After two washes, cells were incubated for 30 min with T4 DNA ligase (Thermo) at room temperature with gentle agitation, followed by a 2 h (flow cytometry) or 4 h (mass cytometry) incubation with phi29 DNA polymerase (Thermo) at 30 °C under agitation.

    Techniques: Staining, Blocking Assay, Hybridization, Amplification, Labeling, Mass Cytometry, Real-time Polymerase Chain Reaction

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Article Snippet: These results of CIAP treatments might perhaps give us at least 3 messages: (i) CIAP treatments of linkers A–B could block, but not completely, the ligation of linkers A–B, indicating that some of linkers A–B could still be joined by T4 DNA ligase even after CIAP treatment; (ii) the ligation of linkers A–B could be recovered after CIAP was inactivated at 85°C for 30–90 min, perhaps suggesting that some of linkers A–B could spontaneously delete one or more nucleotide(s) at their 5′-ends and generate some 5′-phosphate ends when the linkers were incubated at 85°C for 30–90 min; and (iii) these results of CIAP treatments again indicated that band 5 in was the ligation products of linkers A–B.

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Article Snippet: These results of CIAP treatments might perhaps give us at least 3 messages: (i) CIAP treatments of linkers A–B could block, but not completely, the ligation of linkers A–B, indicating that some of linkers A–B could still be joined by T4 DNA ligase even after CIAP treatment; (ii) the ligation of linkers A–B could be recovered after CIAP was inactivated at 85°C for 30–90 min, perhaps suggesting that some of linkers A–B could spontaneously delete one or more nucleotide(s) at their 5′-ends and generate some 5′-phosphate ends when the linkers were incubated at 85°C for 30–90 min; and (iii) these results of CIAP treatments again indicated that band 5 in was the ligation products of linkers A–B.

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Article Snippet: These results of CIAP treatments might perhaps give us at least 3 messages: (i) CIAP treatments of linkers A–B could block, but not completely, the ligation of linkers A–B, indicating that some of linkers A–B could still be joined by T4 DNA ligase even after CIAP treatment; (ii) the ligation of linkers A–B could be recovered after CIAP was inactivated at 85°C for 30–90 min, perhaps suggesting that some of linkers A–B could spontaneously delete one or more nucleotide(s) at their 5′-ends and generate some 5′-phosphate ends when the linkers were incubated at 85°C for 30–90 min; and (iii) these results of CIAP treatments again indicated that band 5 in was the ligation products of linkers A–B.

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Article Snippet: These results of CIAP treatments might perhaps give us at least 3 messages: (i) CIAP treatments of linkers A–B could block, but not completely, the ligation of linkers A–B, indicating that some of linkers A–B could still be joined by T4 DNA ligase even after CIAP treatment; (ii) the ligation of linkers A–B could be recovered after CIAP was inactivated at 85°C for 30–90 min, perhaps suggesting that some of linkers A–B could spontaneously delete one or more nucleotide(s) at their 5′-ends and generate some 5′-phosphate ends when the linkers were incubated at 85°C for 30–90 min; and (iii) these results of CIAP treatments again indicated that band 5 in was the ligation products of linkers A–B.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    PCR primers used in the amplification reaction, and their cognate substrates. We amplify both LTR/host junctions and the primers for both ends of the provirus are shown. Panel a . At the top is a drawing of the T-linker, and the first and second linker primers. The blocking group (NH3, C6) on the 3′ end of the short strand of the T-linker is shown, and the 5′ end of short strand is phosphorylated (P), to allow it to be ligated to the host DNA. The second linker primer is used to add the sequences needed for Illumina sequencing to one end of the amplified fragment. The first and second LTR primers used to amplify the 5′ LTR are shown in the drawing, with the linker primers used to amplify the host-virus DNA junction. The sequences needed for Illumina sequencing to the other end of the amplified fragments are added as part of the second LTR primer. Panel b shows the amplification of the 3′ LTR and the appended host sequences, together with the first and second LTR primers (see also Fig. 2 ). Panel c At the bottom of the drawing, are the corresponding primers used to amplify the 5′ LTR

    Journal: BMC Genomics

    Article Title: An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

    doi: 10.1186/s12864-020-6647-4

    Figure Lengend Snippet: PCR primers used in the amplification reaction, and their cognate substrates. We amplify both LTR/host junctions and the primers for both ends of the provirus are shown. Panel a . At the top is a drawing of the T-linker, and the first and second linker primers. The blocking group (NH3, C6) on the 3′ end of the short strand of the T-linker is shown, and the 5′ end of short strand is phosphorylated (P), to allow it to be ligated to the host DNA. The second linker primer is used to add the sequences needed for Illumina sequencing to one end of the amplified fragment. The first and second LTR primers used to amplify the 5′ LTR are shown in the drawing, with the linker primers used to amplify the host-virus DNA junction. The sequences needed for Illumina sequencing to the other end of the amplified fragments are added as part of the second LTR primer. Panel b shows the amplification of the 3′ LTR and the appended host sequences, together with the first and second LTR primers (see also Fig. 2 ). Panel c At the bottom of the drawing, are the corresponding primers used to amplify the 5′ LTR

    Article Snippet: DNA fragmentation, end repair, and linker ligation We use the NEB Next Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Cat No. E7805S) for this step.

    Techniques: Polymerase Chain Reaction, Amplification, Blocking Assay, Sequencing

    Overview of the linker-mediated amplification of HIV integration sites and the bioinformatic pipeline used to distinguish real integration sites from artifacts. A schematic drawing of an integrated HIV provirus is shown at the top of the figure. As described in the text, and in the Supplementary material, the host DNA is fragmented, the ends are made blunt, and a single dA is added to the 3′ ends of the fragmented host DNA. The T-linker is composed of a long and a short oligonucleotide. The 3′ end of the short oligonucleotide is blocked (marked by an asterisk, see text) to prevent it from being extended in the PCR steps (see Figs. 2 and 3 for the design and sequences of the linker and primers). The UMI, which is shown in yellow, is a random sequence that is used to help determine if two similar amplified segments do (or do not) originate from two different starting pieces of host DNA. In the example shown in the figure (middle of the drawing), next to T-linkers, there are two fragments that contain a host-virus DNA junction that come from a clone of expanded cells. The two fragments have exactly the same integration site, but there are different breakpoints in the host DNA. As described in the text, the PCR reaction must be initiated from the LTR primer because the segment that the first T-linker primer anneals to is not present in the short oligonucleotide of the T-linker. The bottom of the drawing shows a schematic diagram of the amplified DNA and the location of the Illumina Read 1 and Read 2 primers. The Illumina data are processed, using the pipeline, as indicated in the diagram, and the output is saved as an Excel file (the screen shot shows a small portion of an output file)

    Journal: BMC Genomics

    Article Title: An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

    doi: 10.1186/s12864-020-6647-4

    Figure Lengend Snippet: Overview of the linker-mediated amplification of HIV integration sites and the bioinformatic pipeline used to distinguish real integration sites from artifacts. A schematic drawing of an integrated HIV provirus is shown at the top of the figure. As described in the text, and in the Supplementary material, the host DNA is fragmented, the ends are made blunt, and a single dA is added to the 3′ ends of the fragmented host DNA. The T-linker is composed of a long and a short oligonucleotide. The 3′ end of the short oligonucleotide is blocked (marked by an asterisk, see text) to prevent it from being extended in the PCR steps (see Figs. 2 and 3 for the design and sequences of the linker and primers). The UMI, which is shown in yellow, is a random sequence that is used to help determine if two similar amplified segments do (or do not) originate from two different starting pieces of host DNA. In the example shown in the figure (middle of the drawing), next to T-linkers, there are two fragments that contain a host-virus DNA junction that come from a clone of expanded cells. The two fragments have exactly the same integration site, but there are different breakpoints in the host DNA. As described in the text, the PCR reaction must be initiated from the LTR primer because the segment that the first T-linker primer anneals to is not present in the short oligonucleotide of the T-linker. The bottom of the drawing shows a schematic diagram of the amplified DNA and the location of the Illumina Read 1 and Read 2 primers. The Illumina data are processed, using the pipeline, as indicated in the diagram, and the output is saved as an Excel file (the screen shot shows a small portion of an output file)

    Article Snippet: DNA fragmentation, end repair, and linker ligation We use the NEB Next Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Cat No. E7805S) for this step.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing

    Properties of the cleavage activity. RNA substrates and cleavage sites are indicated at the left-hand side of gels. A, Cleavage of STS and its 3′-end extension variants. The total lengths are the STS plus the indicated numbers of nts on the top

    Journal: Plant Physiology

    Article Title: A Novel Plant in Vitro Assay System for Pre-mRNA Cleavage during 3?-End Formation 1A Novel Plant in Vitro Assay System for Pre-mRNA Cleavage during 3?-End Formation 1 [OA]

    doi: 10.1104/pp.111.179465

    Figure Lengend Snippet: Properties of the cleavage activity. RNA substrates and cleavage sites are indicated at the left-hand side of gels. A, Cleavage of STS and its 3′-end extension variants. The total lengths are the STS plus the indicated numbers of nts on the top

    Article Snippet: Cleavage products of cold RNA were gel purified, and a 3′-end RNA linker (miRCat33; Integrated DNA Technology, Inc.) with a preactivated 5′ end and a blocked 3′ end was ligated to the 3′ end of the purified cleavage product, as described by the manufacturer.

    Techniques: Activity Assay