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  • 99
    Thermo Fisher m mlv rt
    Biochemical identification of a basal L1 RNP complex. A. L1 RNPs contain ORF1p, ORF2p, L1 RNA, and L1 reverse transcriptase activity: RNP pellets were obtained from untransfected HeLa cells, or from HeLa cells transfected with wild-type (pAD2TE1) and reverse transcriptase deficient (pAD135) L1 constructs. As in Figure 2 , tagged ORF1p and ORF2p were detected using anti-T7 (αT7) and anti-TAP (αTAP) respectively. Ribosomal S6 protein was detected using an anti-S6 (αS6) antibody and was used as an RNP loading control. Reverse transcriptase activity was detected using the <t>LEAP</t> assay as described previously [17] . Reactions without template (No Template) or RNPs (No RNP/RNA) were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with <t>M-MLV</t> reverse transcriptase control for the presence of L1 RNA in RNPs (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). B. Flow chart of the L1 RNP immunoprecipitation reaction: Whole cell extracts were prepared from HeLa cells transfected with either pDK101 or pES2TE1. Immunoprecipitation reactions were conducted by incubating the resultant lysates with agarose beads fused to an anti-FLAG M2 antibody. The elution of ORF2p from the beads was performed by FLAG peptide competition. Western blotting and LEAP assays were performed on aliquots of the whole cell extracts or the elution fractions to detect the L1-encoded proteins and L1-specific reverse transcriptase activity, respectively. C. Co-immunoprecipitation of ORF1p and ORF2p: Whole cell extract (input) and immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells were subjected to western blotting to identify ORF2p (αHA; top panel), ORF1p (αT7 middle panel) or tubulin (αTubulin, bottom panel). The femto ECL substrate (Pierce) was used to detect ORF1p and ORF2p. D. A basal L1 RNP complex contains L1 RNA and retains L1 reverse transcriptase specific activity: LEAP was performed on whole cell extracts (input) or immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells. Reactions conducted without template (No Template) or without RNPs (No RNP) were used as negative controls. As in Figure 2 , colored cartoons of the constructs are indicated in panels A, C and D. Molecular weight/DNA size markers (Invitrogen) are indicated at the left of the images. All constructs contain the mneoI retrotransposition indicator cassette.
    M Mlv Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1402 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega m mlv rt
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 3381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Evrogen mmlv rt kit
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    Mmlv Rt Kit, supplied by Evrogen, used in various techniques. Bioz Stars score: 92/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher m mlv rt kit
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega m mlv rt kit
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt Kit, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher m mlv rt enzyme
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega m mlv rt system
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt System, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega mmlv rt enzyme
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    Mmlv Rt Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega moloney murine leukemia virus m mlv rt
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    Moloney Murine Leukemia Virus M Mlv Rt, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega mmlv rt buffer
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    Mmlv Rt Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega m mlv rt reaction buffer
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    M Mlv Rt Reaction Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher moloney murine leukemia virus m mlv rt
    Exogenous reverse transcription reactions with MN-treated pFOXC RT and <t>MMLV</t> RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following <t>cDNA</t> synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
    Moloney Murine Leukemia Virus M Mlv Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Biochemical identification of a basal L1 RNP complex. A. L1 RNPs contain ORF1p, ORF2p, L1 RNA, and L1 reverse transcriptase activity: RNP pellets were obtained from untransfected HeLa cells, or from HeLa cells transfected with wild-type (pAD2TE1) and reverse transcriptase deficient (pAD135) L1 constructs. As in Figure 2 , tagged ORF1p and ORF2p were detected using anti-T7 (αT7) and anti-TAP (αTAP) respectively. Ribosomal S6 protein was detected using an anti-S6 (αS6) antibody and was used as an RNP loading control. Reverse transcriptase activity was detected using the LEAP assay as described previously [17] . Reactions without template (No Template) or RNPs (No RNP/RNA) were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in RNPs (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). B. Flow chart of the L1 RNP immunoprecipitation reaction: Whole cell extracts were prepared from HeLa cells transfected with either pDK101 or pES2TE1. Immunoprecipitation reactions were conducted by incubating the resultant lysates with agarose beads fused to an anti-FLAG M2 antibody. The elution of ORF2p from the beads was performed by FLAG peptide competition. Western blotting and LEAP assays were performed on aliquots of the whole cell extracts or the elution fractions to detect the L1-encoded proteins and L1-specific reverse transcriptase activity, respectively. C. Co-immunoprecipitation of ORF1p and ORF2p: Whole cell extract (input) and immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells were subjected to western blotting to identify ORF2p (αHA; top panel), ORF1p (αT7 middle panel) or tubulin (αTubulin, bottom panel). The femto ECL substrate (Pierce) was used to detect ORF1p and ORF2p. D. A basal L1 RNP complex contains L1 RNA and retains L1 reverse transcriptase specific activity: LEAP was performed on whole cell extracts (input) or immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells. Reactions conducted without template (No Template) or without RNPs (No RNP) were used as negative controls. As in Figure 2 , colored cartoons of the constructs are indicated in panels A, C and D. Molecular weight/DNA size markers (Invitrogen) are indicated at the left of the images. All constructs contain the mneoI retrotransposition indicator cassette.

    Journal: PLoS Genetics

    Article Title: Characterization of LINE-1 Ribonucleoprotein Particles

    doi: 10.1371/journal.pgen.1001150

    Figure Lengend Snippet: Biochemical identification of a basal L1 RNP complex. A. L1 RNPs contain ORF1p, ORF2p, L1 RNA, and L1 reverse transcriptase activity: RNP pellets were obtained from untransfected HeLa cells, or from HeLa cells transfected with wild-type (pAD2TE1) and reverse transcriptase deficient (pAD135) L1 constructs. As in Figure 2 , tagged ORF1p and ORF2p were detected using anti-T7 (αT7) and anti-TAP (αTAP) respectively. Ribosomal S6 protein was detected using an anti-S6 (αS6) antibody and was used as an RNP loading control. Reverse transcriptase activity was detected using the LEAP assay as described previously [17] . Reactions without template (No Template) or RNPs (No RNP/RNA) were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in RNPs (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). B. Flow chart of the L1 RNP immunoprecipitation reaction: Whole cell extracts were prepared from HeLa cells transfected with either pDK101 or pES2TE1. Immunoprecipitation reactions were conducted by incubating the resultant lysates with agarose beads fused to an anti-FLAG M2 antibody. The elution of ORF2p from the beads was performed by FLAG peptide competition. Western blotting and LEAP assays were performed on aliquots of the whole cell extracts or the elution fractions to detect the L1-encoded proteins and L1-specific reverse transcriptase activity, respectively. C. Co-immunoprecipitation of ORF1p and ORF2p: Whole cell extract (input) and immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells were subjected to western blotting to identify ORF2p (αHA; top panel), ORF1p (αT7 middle panel) or tubulin (αTubulin, bottom panel). The femto ECL substrate (Pierce) was used to detect ORF1p and ORF2p. D. A basal L1 RNP complex contains L1 RNA and retains L1 reverse transcriptase specific activity: LEAP was performed on whole cell extracts (input) or immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells. Reactions conducted without template (No Template) or without RNPs (No RNP) were used as negative controls. As in Figure 2 , colored cartoons of the constructs are indicated in panels A, C and D. Molecular weight/DNA size markers (Invitrogen) are indicated at the left of the images. All constructs contain the mneoI retrotransposition indicator cassette.

    Article Snippet: For analysis, 1 µL of LEAP or M-MLV RT products was added to 19 µL of master mix (1X SYBR Green PCR Master Mix (Applied Biosystems), 500 nM L1 3′ end primer, and 500 nM L1 Reverse primer), and amplified in a standard Q-PCR run of 45 cycles.

    Techniques: Activity Assay, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunoprecipitation, Western Blot, Molecular Weight

    TAP tagged ORF2p and RT activity detection in RNP preparation. A. Schematic representation of the amino acid mutation positions in L1 sequence: The names of plasmids containing L1s with mutations in the ORF1p coiled-coil domain (CC-LZ), the ORF1p RNA recognition motif (RRM), and the ORF1p carboxyl-terminal (CTD) domain are indicated below the schematic. The names of plasmids containing mutations in the ORF2p endonuclease domain (EN), reverse transcriptase domain (RT) or cysteine-rich domain (C) also are shown. pADL/R is a double mutant that contains a putative leucine zipper mutation and a carboxyl-terminal domain mutation in ORF1p. pADL/C is a double mutant that contains a putative leucine zipper mutation in ORF1p and a C-domain mutation in ORF2p. The flags indicate the epitope tag present on ORF1 and ORF2. B. Detection of ORF1p and ORF2p from mutant L1 constructs: RNPs from HeLa cells transfected with a RC-L1 (pAD2TE1) or the indicated mutant L1 constructs (see Figure 4A ) were analyzed by western blotting [16] . Tagged L1 proteins were detected as in Figure 3 ; ORF2p (top panel), ORF1p (middle panel). Ribosomal S6 protein detection was used as a loading control (bottom panel). Molecular weight markers (Invitrogen) are indicated at the left of the image. C. L1 RT activity of RNP fractions detected by LEAP: An aliquot from each of the indicated RNP preparations noted above was used to perform LEAP assays (see Figure 3 ) [17] . RNPs from pAD2TE1 served as a positive control. RNPs from untransfected HeLa cells or pAD135 (D 702 A; RT mutant) transfected cells served as negative controls. Reactions without RNPs (No RNP/RNA) or template (No Template) also were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in the RNP fractions (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). DNA size markers (Invitrogen) are indicated at the left of the image. All constructs in panel B and C contain the mneoI retrotransposition indicator cassette.

    Journal: PLoS Genetics

    Article Title: Characterization of LINE-1 Ribonucleoprotein Particles

    doi: 10.1371/journal.pgen.1001150

    Figure Lengend Snippet: TAP tagged ORF2p and RT activity detection in RNP preparation. A. Schematic representation of the amino acid mutation positions in L1 sequence: The names of plasmids containing L1s with mutations in the ORF1p coiled-coil domain (CC-LZ), the ORF1p RNA recognition motif (RRM), and the ORF1p carboxyl-terminal (CTD) domain are indicated below the schematic. The names of plasmids containing mutations in the ORF2p endonuclease domain (EN), reverse transcriptase domain (RT) or cysteine-rich domain (C) also are shown. pADL/R is a double mutant that contains a putative leucine zipper mutation and a carboxyl-terminal domain mutation in ORF1p. pADL/C is a double mutant that contains a putative leucine zipper mutation in ORF1p and a C-domain mutation in ORF2p. The flags indicate the epitope tag present on ORF1 and ORF2. B. Detection of ORF1p and ORF2p from mutant L1 constructs: RNPs from HeLa cells transfected with a RC-L1 (pAD2TE1) or the indicated mutant L1 constructs (see Figure 4A ) were analyzed by western blotting [16] . Tagged L1 proteins were detected as in Figure 3 ; ORF2p (top panel), ORF1p (middle panel). Ribosomal S6 protein detection was used as a loading control (bottom panel). Molecular weight markers (Invitrogen) are indicated at the left of the image. C. L1 RT activity of RNP fractions detected by LEAP: An aliquot from each of the indicated RNP preparations noted above was used to perform LEAP assays (see Figure 3 ) [17] . RNPs from pAD2TE1 served as a positive control. RNPs from untransfected HeLa cells or pAD135 (D 702 A; RT mutant) transfected cells served as negative controls. Reactions without RNPs (No RNP/RNA) or template (No Template) also were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in the RNP fractions (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). DNA size markers (Invitrogen) are indicated at the left of the image. All constructs in panel B and C contain the mneoI retrotransposition indicator cassette.

    Article Snippet: For analysis, 1 µL of LEAP or M-MLV RT products was added to 19 µL of master mix (1X SYBR Green PCR Master Mix (Applied Biosystems), 500 nM L1 3′ end primer, and 500 nM L1 Reverse primer), and amplified in a standard Q-PCR run of 45 cycles.

    Techniques: Activity Assay, Mutagenesis, Sequencing, Construct, Transfection, Western Blot, Molecular Weight, Positive Control, Reverse Transcription Polymerase Chain Reaction

    Analysis of MMLV integrations in the Hoxa locus of E2a–PBX1 B-cell leukemia. ( A ) Schematic representation of MMLV integrations localized in the Hoxa locus in E2a–PBX1 transgenic mice as determined by I-PCR. Integrations are indicated by the vertical arrows above the representation of the Hoxa cluster. Large vertical bars indicate Hoxa gene exons, small vertical bars indicate the UTRs, and solid lines with arrows pointing to the left indicate the orientation of the gene and the introns. ( B ) Table showing Q-PCR results as fold difference in expression levels of Hoxa cluster genes in the Hoxa -locus-targeted tumors compared to a control non- Hoxa ), values of 8, representing three PCR cycles, and more are considered as substantial increases of expression (boxed numbers). ( C ) Semiquantitative RT–PCR analyses of Hoxa7, Hoxa9 ,and Hoxa10 expression in E2a–PBX1 B-cell leukemias that contain MMLV integration(s) in the Hoxa locus (lanes 5 – 10 ) or control B-cell leukemias (lanes 3,4 ). (S) RNA extracted from spleen cells of a healthy E2a–PBX1 transgenic mouse; (no RT) no reverse transcription before the PCR amplification.

    Journal: Genes & Development

    Article Title: High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia

    doi: 10.1101/gad.1268505

    Figure Lengend Snippet: Analysis of MMLV integrations in the Hoxa locus of E2a–PBX1 B-cell leukemia. ( A ) Schematic representation of MMLV integrations localized in the Hoxa locus in E2a–PBX1 transgenic mice as determined by I-PCR. Integrations are indicated by the vertical arrows above the representation of the Hoxa cluster. Large vertical bars indicate Hoxa gene exons, small vertical bars indicate the UTRs, and solid lines with arrows pointing to the left indicate the orientation of the gene and the introns. ( B ) Table showing Q-PCR results as fold difference in expression levels of Hoxa cluster genes in the Hoxa -locus-targeted tumors compared to a control non- Hoxa ), values of 8, representing three PCR cycles, and more are considered as substantial increases of expression (boxed numbers). ( C ) Semiquantitative RT–PCR analyses of Hoxa7, Hoxa9 ,and Hoxa10 expression in E2a–PBX1 B-cell leukemias that contain MMLV integration(s) in the Hoxa locus (lanes 5 – 10 ) or control B-cell leukemias (lanes 3,4 ). (S) RNA extracted from spleen cells of a healthy E2a–PBX1 transgenic mouse; (no RT) no reverse transcription before the PCR amplification.

    Article Snippet: Total RNA was isolated by Trizol, DNase-I-treated, and cDNA was prepared (MMLV-RT, random primers) according to the manufacturer's instructions (InVitrogen).

    Techniques: Transgenic Assay, Mouse Assay, Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    Exogenous reverse transcription reactions with MN-treated pFOXC RT and MMLV RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following cDNA synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.

    Journal: Eukaryotic Cell

    Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum

    doi: 10.1128/EC.3.6.1589-1600.2004

    Figure Lengend Snippet: Exogenous reverse transcription reactions with MN-treated pFOXC RT and MMLV RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following cDNA synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.

    Article Snippet: Interestingly, the major cDNA products obtained with MMLV RT were significantly shorter (73, 76, and 82 nt) than those obtained with the MN-treated pFOXC RT.

    Techniques: Isolation, Plasmid Preparation, Incubation, Electrophoresis, Marker, Molecular Weight, Labeling