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  • 99
    New England Biolabs kpni
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Kpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher kpni fragment
    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, <t>BamHI</t> and <t>KpnI</t> sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.
    Kpni Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher kpni
    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major <t>LMSTI1</t> and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with <t>KpnI</t> and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    Kpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fastdigest kpni
    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major <t>LMSTI1</t> and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with <t>KpnI</t> and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    Fastdigest Kpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs kpni hf
    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a <t>Kpn</t> I restriction site used for genotyping of the ter allele performing <t>PCR</t> amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.
    Kpni Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jena Bioscience kpni restriction enzymes
    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a <t>Kpn</t> I restriction site used for genotyping of the ter allele performing <t>PCR</t> amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.
    Kpni Restriction Enzymes, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega kpni
    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a <t>Kpn</t> I restriction site used for genotyping of the ter allele performing <t>PCR</t> amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.
    Kpni, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4043 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kaneka Corp kpni
    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a <t>Kpn</t> I restriction site used for genotyping of the ter allele performing <t>PCR</t> amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.
    Kpni, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    kpni  (Roche)
    93
    Roche kpni
    Mutation of the furin cleavage site (FCS) of MuHV-4 gB. (A) The location of the FCS within a structure model of the MuHV-4 gB ectodomain (amino acids 60 to 680) is shown in purple. The amino acid and nucleotide sequences of the wild-type (WT) FCS and the ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutants are shown. A diagnostic <t>KpnI</t> restriction site was introduced in the ΔFCSv2 and ΔFCSv3 mutants. The site at which furin is expected to cleave gB is indicated by an arrow. I to V, domains I to V; N, amino terminus; C, carboxy terminus; FL, fusion loops. (B) Schematic drawing of the MuHV-4 ORF8 encoding gB. HindIII and KpnI restriction sites, the HindIII fragment used as probe for Southern blot hybridization, and the position of primers P1 and P2 used for PCR analysis are shown. (C) Restriction enzyme analysis of mutant MuHV-4 BACs. The WT BAC and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant BACs were digested with HindIII and separated by agarose gel electrophoresis. (D) Southern blot analysis of mutant viruses. Viral <t>DNA</t> was extracted, digested with KpnI, and analyzed by Southern blotting hybridization with the radioactively labeled MuHV-4 HindIII N fragment. In the mutant viruses ΔFCSv2 and ΔFCSv3, the 5.6-kb KpnI fragment is cleaved into 3.2-kb and 2.4-kb bands due to the introduced KpnI site. (E) PCR analysis of mutant viruses. Viral DNA was subjected to PCR with primers P1 and P2 spanning the sequence encoding the FCS, followed by agarose gel electrophoresis. The observed PCR products correspond to the expected sizes shown below the gel image (in bp).
    Kpni, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore kpni
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa kpni bgiii
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Bgiii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Promega kpni bglii
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Bglii, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega kpni xhoi
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Xhoi, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega kpni digestion
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Digestion, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche kpni nhei
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Nhei, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche kpni asp718i
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Asp718i, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega kpni bgiii
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Bgiii, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa kpni kpni fragments
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni Kpni Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Promega restriction enzyme kpni
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Restriction Enzyme Kpni, supplied by Promega, used in various techniques. Bioz Stars score: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Stratagene kpni
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim kpni
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
    Kpni, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare kpni
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
    Kpni, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    kpni  (Lonza)
    92
    Lonza kpni
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
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    Bio Basic Canada kpni restriction sites
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
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    85
    GenScript kpni restriction sites
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
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    88
    Promega kpni restriction sites
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
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    94
    Genechem kpni
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
    Kpni, supplied by Genechem, used in various techniques. Bioz Stars score: 94/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Epicentre Biotechnologies kpni
    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). <t>DNA</t> from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with <t>KpnI,</t> an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.
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    Image Search Results


    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: In Vitro, Selection

    Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Quantitation Assay, Binding Assay, Mobility Shift

    Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Titration, Concentration Assay

    Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Binding Assay, Mobility Shift

    Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Activity Assay, Mobility Shift, Binding Assay, Inhibition, Incubation, Concentration Assay

    Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Binding Assay

    ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques:

    KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Incubation, Concentration Assay

    Restriction digestion patterns of PERV clones. A , Five patterns (A-E) of MboI digestion and B , four patterns (a-d) of KpnI digestion were identified in the PCR product amplified by Easy A polymerase high fidelity.

    Journal: Virology Journal

    Article Title: Characterization of PERV in a new conserved pig herd as potential donor animals for xenotransplantation in China

    doi: 10.1186/s12985-014-0212-1

    Figure Lengend Snippet: Restriction digestion patterns of PERV clones. A , Five patterns (A-E) of MboI digestion and B , four patterns (a-d) of KpnI digestion were identified in the PCR product amplified by Easy A polymerase high fidelity.

    Article Snippet: Three hundred single positive clones were picked for PCR amplification, and the PCR products were analyzed using the restriction enzymes KpnI and MboI (New England Biolabs, MA, USA).

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification

    Southern blot and PCR analysis for wt AAV DNA at the site of administration. (A) Southern blot of unamplified, Kpn I-digested genomic DNA isolated from the site of wt AAV administration. Liver tissue from animals infected intravenously (lanes 1 and 2), muscle tissue from animals infected intramuscularly (lanes 4 to 6), and nasal tissue from animals infected intranasally either without (lanes 8 and 9) or with (lanes 10 and 11) helper Ad were examined. Liver, muscle, and nasal epithelial DNAs from a saline-injected control animal were also examined (lanes 3, 7, and 12, respectively). Plasmid DNAs equating to 0.1, 1, 10, and 100 copies of wt AAV genomes per cell were included as standards (lanes 13 to 16, respectively). (B) PCR analysis for internal AAV-Rep gene sequences was performed for each of the same samples.

    Journal: Journal of Virology

    Article Title: Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model

    doi:

    Figure Lengend Snippet: Southern blot and PCR analysis for wt AAV DNA at the site of administration. (A) Southern blot of unamplified, Kpn I-digested genomic DNA isolated from the site of wt AAV administration. Liver tissue from animals infected intravenously (lanes 1 and 2), muscle tissue from animals infected intramuscularly (lanes 4 to 6), and nasal tissue from animals infected intranasally either without (lanes 8 and 9) or with (lanes 10 and 11) helper Ad were examined. Liver, muscle, and nasal epithelial DNAs from a saline-injected control animal were also examined (lanes 3, 7, and 12, respectively). Plasmid DNAs equating to 0.1, 1, 10, and 100 copies of wt AAV genomes per cell were included as standards (lanes 13 to 16, respectively). (B) PCR analysis for internal AAV-Rep gene sequences was performed for each of the same samples.

    Article Snippet: The DNA (30 μg) was digested for 24 h with Kpn I (New England BioLabs) under conditions recommended by the manufacturer.

    Techniques: Southern Blot, Polymerase Chain Reaction, Isolation, Infection, Injection, Plasmid Preparation

    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Generated, Construct, Expressing, Injection, Fluorescence In Situ Hybridization, Fluorescence, Activity Assay

    Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Plasmid Preparation, Clone Assay, TA Cloning, Ligation

    End configuration of the DNA substrates used in this study. The diagram presents the DNA ends created by restriction of M13 mp18 derivatives with Xma I and Mlu I (XM), Sma I (S) or Aat II and Kpn I (AK). Sites of homology to the protruding DNA ends are shown in bold.

    Journal: Nucleic Acids Research

    Article Title: The role of DNA polymerase activity in human non-homologous end joining

    doi:

    Figure Lengend Snippet: End configuration of the DNA substrates used in this study. The diagram presents the DNA ends created by restriction of M13 mp18 derivatives with Xma I and Mlu I (XM), Sma I (S) or Aat II and Kpn I (AK). Sites of homology to the protruding DNA ends are shown in bold.

    Article Snippet: The DNA was digested with Sma I (Pharmacia), with Kpn I and Aat II or sequentially with Xma I (Pharmacia) or its schizoisomer Cfr 9I (MBI Fermentas) and Mlu I (Pharmacia) to give the substrates S, AK and XM, respectively (Fig. ).

    Techniques:

    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Journal: Reports of Biochemistry & Molecular Biology

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi

    doi:

    Figure Lengend Snippet: A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Article Snippet: Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer.

    Techniques: Construct, Expressing, Plasmid Preparation

    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Journal: PLoS ONE

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

    doi: 10.1371/journal.pone.0038001

    Figure Lengend Snippet: Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Article Snippet: The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control

    Mutation of the furin cleavage site (FCS) of MuHV-4 gB. (A) The location of the FCS within a structure model of the MuHV-4 gB ectodomain (amino acids 60 to 680) is shown in purple. The amino acid and nucleotide sequences of the wild-type (WT) FCS and the ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutants are shown. A diagnostic KpnI restriction site was introduced in the ΔFCSv2 and ΔFCSv3 mutants. The site at which furin is expected to cleave gB is indicated by an arrow. I to V, domains I to V; N, amino terminus; C, carboxy terminus; FL, fusion loops. (B) Schematic drawing of the MuHV-4 ORF8 encoding gB. HindIII and KpnI restriction sites, the HindIII fragment used as probe for Southern blot hybridization, and the position of primers P1 and P2 used for PCR analysis are shown. (C) Restriction enzyme analysis of mutant MuHV-4 BACs. The WT BAC and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant BACs were digested with HindIII and separated by agarose gel electrophoresis. (D) Southern blot analysis of mutant viruses. Viral DNA was extracted, digested with KpnI, and analyzed by Southern blotting hybridization with the radioactively labeled MuHV-4 HindIII N fragment. In the mutant viruses ΔFCSv2 and ΔFCSv3, the 5.6-kb KpnI fragment is cleaved into 3.2-kb and 2.4-kb bands due to the introduced KpnI site. (E) PCR analysis of mutant viruses. Viral DNA was subjected to PCR with primers P1 and P2 spanning the sequence encoding the FCS, followed by agarose gel electrophoresis. The observed PCR products correspond to the expected sizes shown below the gel image (in bp).

    Journal: Journal of Virology

    Article Title: Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells

    doi: 10.1128/JVI.00709-13

    Figure Lengend Snippet: Mutation of the furin cleavage site (FCS) of MuHV-4 gB. (A) The location of the FCS within a structure model of the MuHV-4 gB ectodomain (amino acids 60 to 680) is shown in purple. The amino acid and nucleotide sequences of the wild-type (WT) FCS and the ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutants are shown. A diagnostic KpnI restriction site was introduced in the ΔFCSv2 and ΔFCSv3 mutants. The site at which furin is expected to cleave gB is indicated by an arrow. I to V, domains I to V; N, amino terminus; C, carboxy terminus; FL, fusion loops. (B) Schematic drawing of the MuHV-4 ORF8 encoding gB. HindIII and KpnI restriction sites, the HindIII fragment used as probe for Southern blot hybridization, and the position of primers P1 and P2 used for PCR analysis are shown. (C) Restriction enzyme analysis of mutant MuHV-4 BACs. The WT BAC and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant BACs were digested with HindIII and separated by agarose gel electrophoresis. (D) Southern blot analysis of mutant viruses. Viral DNA was extracted, digested with KpnI, and analyzed by Southern blotting hybridization with the radioactively labeled MuHV-4 HindIII N fragment. In the mutant viruses ΔFCSv2 and ΔFCSv3, the 5.6-kb KpnI fragment is cleaved into 3.2-kb and 2.4-kb bands due to the introduced KpnI site. (E) PCR analysis of mutant viruses. Viral DNA was subjected to PCR with primers P1 and P2 spanning the sequence encoding the FCS, followed by agarose gel electrophoresis. The observed PCR products correspond to the expected sizes shown below the gel image (in bp).

    Article Snippet: For the latter, viral DNA was digested with KpnI (Roche), separated on a 1% agarose gel, transferred and UV-cross-linked to a positively charged nylon membrane (Roche), hybridized with a P32-labeled probe consisting of the MuHV-4 genomic HindIII N fragment (genomic coordinates 16239 to 19870), and exposed to X-ray films (Fujifilm).

    Techniques: Mutagenesis, Diagnostic Assay, Southern Blot, Hybridization, Polymerase Chain Reaction, BAC Assay, Agarose Gel Electrophoresis, Labeling, Sequencing

    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites KpnI and Xhol ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE

    Journal: Cell Stress & Chaperones

    Article Title: Genetic polymorphism in Hsp90AA1 gene is associated with the thermotolerance in Chinese Holstein cows

    doi: 10.1007/s12192-017-0873-y

    Figure Lengend Snippet: A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites KpnI and Xhol ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE

    Article Snippet: To explore whether the g.− 87 G > C SNP could affect the promoter activity of the bovine Hsp90AA1 , 296 bp of genomic DNA between − 237 and + 59 of each wild-type allele G and mutant allele C were cloned into a promoterless pGL3-basic vector (Promega, USA) KpnI and Xhol restriction sites (Fig. a, b) to construct pGL3-G308 and pGL3-C308 plasmids, which were subsequently transferred into Hela (Sigma USA).

    Techniques: Plasmid Preparation, Negative Control, Transfection, Luciferase, Activity Assay, Expressing, Mutagenesis

    Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). DNA from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with KpnI, an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.

    Journal: Journal of Clinical Investigation

    Article Title: Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

    doi:

    Figure Lengend Snippet: Analysis of engraftment with transduced cells in vivo. ( a ) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). DNA from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H 2 O: reagent control. ( b ) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with KpnI, an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.

    Article Snippet: Ten micrograms of genomic DNA was digested with KpnI (Boehringer Mannheim Biochemicals Inc., Indianapolis, Indiana, USA), which cuts once within each viral LTR and thus can distinguish integrated retroviral G1Na and LNL6 DNA.

    Techniques: In Vivo, Polymerase Chain Reaction, Transduction, Plasmid Preparation, Transplantation Assay, Southern Blot