Structured Review

Promega pcr mix
Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase <t>PCR,</t> using primers from 5 BLV genome regions, was used to amplify products from <t>DNA</t> extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Pcr Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Bovine Leukemia Virus DNA in Human Breast Tissue"

Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

Journal: Emerging Infectious Diseases

doi: 10.3201/eid2005.131298

Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Figure Legend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.
Figure Legend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

2) Product Images from "OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics"

Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

Journal: Journal of Immunology Research

doi: 10.1155/2018/2505818

Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
Figure Legend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

3) Product Images from "Histone deacetylase inhibitors induce the expression of tumor suppressor genes Per1 and Per2 in human gastric cancer cells"

Article Title: Histone deacetylase inhibitors induce the expression of tumor suppressor genes Per1 and Per2 in human gastric cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2018.8851

Methylation specific PCR to analyze the methylation status of Per1 and Per2 promoters. KATO III cells were treated with 3 mM NaB, 100 nM TSA for 48 h or 5 mM Aza for 96 h. Then DNA was subjected to bisulfite modification and PCR was conducted with specific primers to distinguish methylated (Lane M) and unmethylated (Lane U) forms of CpGs at the Per1 and Per2 promoters. PCR products were separated on 2% agarose gels. (A) MS-PCR of untreated control KATO III cells. (B) MS-PCR of NaB, TSA or Aza treated KATO III cells. (C) Reverse transcription-quantitative PCR showing upregulation of Per2 mRNA in Aza treated KATO III cells. ***P
Figure Legend Snippet: Methylation specific PCR to analyze the methylation status of Per1 and Per2 promoters. KATO III cells were treated with 3 mM NaB, 100 nM TSA for 48 h or 5 mM Aza for 96 h. Then DNA was subjected to bisulfite modification and PCR was conducted with specific primers to distinguish methylated (Lane M) and unmethylated (Lane U) forms of CpGs at the Per1 and Per2 promoters. PCR products were separated on 2% agarose gels. (A) MS-PCR of untreated control KATO III cells. (B) MS-PCR of NaB, TSA or Aza treated KATO III cells. (C) Reverse transcription-quantitative PCR showing upregulation of Per2 mRNA in Aza treated KATO III cells. ***P

Techniques Used: Methylation, Polymerase Chain Reaction, Modification, Mass Spectrometry, Real-time Polymerase Chain Reaction

4) Product Images from "OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics"

Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

Journal: Journal of Immunology Research

doi: 10.1155/2018/2505818

Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
Figure Legend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

5) Product Images from "Functional Characterization of Synechocystis sp. Strain PCC 6803 pst1 and pst2 Gene Clusters Reveals a Novel Strategy for Phosphate Uptake in a Freshwater Cyanobacterium ▿"

Article Title: Functional Characterization of Synechocystis sp. Strain PCC 6803 pst1 and pst2 Gene Clusters Reveals a Novel Strategy for Phosphate Uptake in a Freshwater Cyanobacterium ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00258-10

PCR determination of the disruption of the targeted genes compared to Synechocystis WT. Data for sphX , pstS1 , and pstS2 (A) and pst (B) gene clusters are shown. This demonstrates that the disruption/deletion was obtained in all cases and that complete
Figure Legend Snippet: PCR determination of the disruption of the targeted genes compared to Synechocystis WT. Data for sphX , pstS1 , and pstS2 (A) and pst (B) gene clusters are shown. This demonstrates that the disruption/deletion was obtained in all cases and that complete

Techniques Used: Polymerase Chain Reaction

6) Product Images from "Differential isoform expression and alternative splicing in sex determination in mice"

Article Title: Differential isoform expression and alternative splicing in sex determination in mice

Journal: BMC Genomics

doi: 10.1186/s12864-019-5572-x

Differentially expressed genes (DEG) in male and female gonads at E11 and E12 and analysis of gene clustering. ( a ) Quantitative PCR analysis of Sry expression in male embryonic day 11 and 12.5 (E11-E12.5) gonads. Biological triplicate results are presented as mean ± SEM. Bars with different superscripts differ significantly (ANOVA, P
Figure Legend Snippet: Differentially expressed genes (DEG) in male and female gonads at E11 and E12 and analysis of gene clustering. ( a ) Quantitative PCR analysis of Sry expression in male embryonic day 11 and 12.5 (E11-E12.5) gonads. Biological triplicate results are presented as mean ± SEM. Bars with different superscripts differ significantly (ANOVA, P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

7) Product Images from "Host-Range Dynamics of Cochliobolus lunatus: From a Biocontrol Agent to a Severe Environmental Threat"

Article Title: Host-Range Dynamics of Cochliobolus lunatus: From a Biocontrol Agent to a Severe Environmental Threat

Journal: BioMed Research International

doi: 10.1155/2014/378372

(a) Agarose gel electrophoresis for PCR products (680 bp) from GPDH locus of Cochliobolus lunatus strains separated on a 2.5% agarose gel. Lane-1 and-7 DNA ladder and lane-2, -3, -4, -5, and -6 are Cochliobolus lunatus strains with DDBJ accessions AB859034, AB859035, AB859036, AB859037, and AB859038, respectively. (b) Molecular phylogenetic analysis by maximum likelihood method based on the TN93 + G + I substitution model [ 22 ]; AIC is 2974.83; BIC is 3449.49; the highest log likelihood is −1494.28 and bootstrap values ≥50% from 1000 iterations are shown. Subcluster (IV) contains strains of Cochliobolus lunatus which causes foliar necrosis of potato.
Figure Legend Snippet: (a) Agarose gel electrophoresis for PCR products (680 bp) from GPDH locus of Cochliobolus lunatus strains separated on a 2.5% agarose gel. Lane-1 and-7 DNA ladder and lane-2, -3, -4, -5, and -6 are Cochliobolus lunatus strains with DDBJ accessions AB859034, AB859035, AB859036, AB859037, and AB859038, respectively. (b) Molecular phylogenetic analysis by maximum likelihood method based on the TN93 + G + I substitution model [ 22 ]; AIC is 2974.83; BIC is 3449.49; the highest log likelihood is −1494.28 and bootstrap values ≥50% from 1000 iterations are shown. Subcluster (IV) contains strains of Cochliobolus lunatus which causes foliar necrosis of potato.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

(a) Archetypal brown-to-black leaf spot disease caused by Cochliobolus lunatus on potato cv. Kufri Jyoti as previously validated by Koch's postulates [ 20 ]. (b) Agarose gel electrophoresis for PCR products (475 bp) from rDNA locus of Cochliobolus lunatus strains separated on a 2.5% agarose gel. Lane-1 DNA ladder and lane-2, -3, -4, -5, and -6 are Cochliobolus lunatus strains with GenBank accessions numbers JX512810, JX512809, JX907827, JX477595, and JX907828, respectively. (c) Molecular phylogenetic analysis by maximum likelihood method based on the K2 + G substitution model [ 21 ]; AIC is 871.49; BIC is 1093.89; the highest log likelihood is −429.87 and bootstrap values ≥50% from 1000 iterations are shown. Subcluster I contain strains of Cochliobolus lunatus which causes foliar necrosis of potato.
Figure Legend Snippet: (a) Archetypal brown-to-black leaf spot disease caused by Cochliobolus lunatus on potato cv. Kufri Jyoti as previously validated by Koch's postulates [ 20 ]. (b) Agarose gel electrophoresis for PCR products (475 bp) from rDNA locus of Cochliobolus lunatus strains separated on a 2.5% agarose gel. Lane-1 DNA ladder and lane-2, -3, -4, -5, and -6 are Cochliobolus lunatus strains with GenBank accessions numbers JX512810, JX512809, JX907827, JX477595, and JX907828, respectively. (c) Molecular phylogenetic analysis by maximum likelihood method based on the K2 + G substitution model [ 21 ]; AIC is 871.49; BIC is 1093.89; the highest log likelihood is −429.87 and bootstrap values ≥50% from 1000 iterations are shown. Subcluster I contain strains of Cochliobolus lunatus which causes foliar necrosis of potato.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

8) Product Images from "DNA typing in thirty seconds with a microfabricated device"

Article Title: DNA typing in thirty seconds with a microfabricated device

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

( a ) Microchip electropherogram for the four-locus CTTv allelic sizing standard. The chip contained 45-μm-deep channels, a 100-μm sample injector, and a 26-mm-long separation channel. The separation was performed at 50°C with a field strength of 200 V/cm in a sieving matrix that consisted of 4% linear polyacrylamide in 1× TBE buffer with 3.5 M urea and 30% (vol/vol) formamide. ( b ) Microchip electropherogram presenting the allelic profile of an individual obtained by spiking a PCR-amplified sample with the CTTv sizing standard. Allele numbers are given above the peaks. Electrophoretic conditions were the same as above.
Figure Legend Snippet: ( a ) Microchip electropherogram for the four-locus CTTv allelic sizing standard. The chip contained 45-μm-deep channels, a 100-μm sample injector, and a 26-mm-long separation channel. The separation was performed at 50°C with a field strength of 200 V/cm in a sieving matrix that consisted of 4% linear polyacrylamide in 1× TBE buffer with 3.5 M urea and 30% (vol/vol) formamide. ( b ) Microchip electropherogram presenting the allelic profile of an individual obtained by spiking a PCR-amplified sample with the CTTv sizing standard. Allele numbers are given above the peaks. Electrophoretic conditions were the same as above.

Techniques Used: MicroChIP Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

9) Product Images from "Bovine Leukemia Virus DNA in Human Breast Tissue"

Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

Journal: Emerging Infectious Diseases

doi: 10.3201/eid2005.131298

Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Figure Legend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.
Figure Legend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

10) Product Images from "Bovine Leukemia Virus DNA in Human Breast Tissue"

Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

Journal: Emerging Infectious Diseases

doi: 10.3201/eid2005.131298

Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Figure Legend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.
Figure Legend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

11) Product Images from "Bovine Leukemia Virus DNA in Human Breast Tissue"

Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

Journal: Emerging Infectious Diseases

doi: 10.3201/eid2005.131298

Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Figure Legend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.
Figure Legend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

12) Product Images from "Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein"

Article Title: Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12203-12210.2003

Analysis of RNA and DNA from Peromyscus cells infected with PCMV. (A) Demonstration of a presence of both genomic and spliced version of P33 gene in infected cells. Lanes: 1, P33-specific RT-PCR with RNA from PCMV-infected cells as a template; 2, P33-specific PCR with purified genomic PCMV DNA as a template; M, 100-bp ladder (Gibco-BRL). (B) Genomic arrangement of coding region of P33. The first exon, encoding the first 11 amino acids, is separated by an 85-bp intron from the second exon encoding an additional 404 amino acids.
Figure Legend Snippet: Analysis of RNA and DNA from Peromyscus cells infected with PCMV. (A) Demonstration of a presence of both genomic and spliced version of P33 gene in infected cells. Lanes: 1, P33-specific RT-PCR with RNA from PCMV-infected cells as a template; 2, P33-specific PCR with purified genomic PCMV DNA as a template; M, 100-bp ladder (Gibco-BRL). (B) Genomic arrangement of coding region of P33. The first exon, encoding the first 11 amino acids, is separated by an 85-bp intron from the second exon encoding an additional 404 amino acids.

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

Southern blot of recombinant and wt-PCMV DNA demonstrating insertion of SNV-G1 into P33. (A) PCR amplification with P33-specific primers with wt-PCMV DNA (lane 1), recombinant PCMV DNA (before FACS) (lane 2), and recombinant DNA (after FACS) (lane 3). (B) Southern blot of total DNA isolated from PMEC infected with PCMV(ΔP33:G1EGFP) (lane 1), wt-PCMV (lane 2), or uninfected control cells (lane 3). The DNAs were cleaved with Bam HI, and the blot was probed with the 1.2-kb Age I fragment containing the P33 gene. (C) Agarose gel, used for the Southern blot presented in panel B. Lane labeling is the same in panels B and C.
Figure Legend Snippet: Southern blot of recombinant and wt-PCMV DNA demonstrating insertion of SNV-G1 into P33. (A) PCR amplification with P33-specific primers with wt-PCMV DNA (lane 1), recombinant PCMV DNA (before FACS) (lane 2), and recombinant DNA (after FACS) (lane 3). (B) Southern blot of total DNA isolated from PMEC infected with PCMV(ΔP33:G1EGFP) (lane 1), wt-PCMV (lane 2), or uninfected control cells (lane 3). The DNAs were cleaved with Bam HI, and the blot was probed with the 1.2-kb Age I fragment containing the P33 gene. (C) Agarose gel, used for the Southern blot presented in panel B. Lane labeling is the same in panels B and C.

Techniques Used: Southern Blot, Recombinant, Polymerase Chain Reaction, Amplification, FACS, Isolation, Infection, Agarose Gel Electrophoresis, Labeling

13) Product Images from "Bovine leukemia virus linked to breast cancer in Australian women and identified before breast cancer development"

Article Title: Bovine leukemia virus linked to breast cancer in Australian women and identified before breast cancer development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0179367

BLV-specific DNA in human breast tissue amplified and detected by PCR-in situ hybridization. A) Breast tissue specimen diagnosed as invasive pleomorphic lobular carcinoma. Three streaks of BLV-positive (brown staining) mammary epithelial cells are seen invading through connective tissue (fibroblasts and adipocytes) that is largely BLV-negative. Positive reactions in mammary epithelial cells are in cytoplasm (diffuse light brown reaction) of most mammary epithelial cells, and nuclei of some cells (diffuse brown reaction as well as dark dots) and as compared to B) Adjacent tissue section without the BLV-specific ISH probe in the hybridization mix. This serves as a background control against which to compare A, and also a control for reactions due to artifacts inherent in some tissues (excess peroxidase and/or melanin). No brown cells are apparent in B. C) Breast tissue specimen diagnosed as benign ductal hyperplasia (normal). Note oval nest of BLV-positive mammary epithelial cells tightly surrounded by fibroblasts that are BLV-negative. Positive reactions in the mammary epithelial cells are intense in the cytoplasm and some cells have evidence of positive nuclear reactions (dark brown dots). D) Background control for C has no apparent brown cells. Light counterstain for all specimens illustrated is Difquick blue and the magnification is x40.
Figure Legend Snippet: BLV-specific DNA in human breast tissue amplified and detected by PCR-in situ hybridization. A) Breast tissue specimen diagnosed as invasive pleomorphic lobular carcinoma. Three streaks of BLV-positive (brown staining) mammary epithelial cells are seen invading through connective tissue (fibroblasts and adipocytes) that is largely BLV-negative. Positive reactions in mammary epithelial cells are in cytoplasm (diffuse light brown reaction) of most mammary epithelial cells, and nuclei of some cells (diffuse brown reaction as well as dark dots) and as compared to B) Adjacent tissue section without the BLV-specific ISH probe in the hybridization mix. This serves as a background control against which to compare A, and also a control for reactions due to artifacts inherent in some tissues (excess peroxidase and/or melanin). No brown cells are apparent in B. C) Breast tissue specimen diagnosed as benign ductal hyperplasia (normal). Note oval nest of BLV-positive mammary epithelial cells tightly surrounded by fibroblasts that are BLV-negative. Positive reactions in the mammary epithelial cells are intense in the cytoplasm and some cells have evidence of positive nuclear reactions (dark brown dots). D) Background control for C has no apparent brown cells. Light counterstain for all specimens illustrated is Difquick blue and the magnification is x40.

Techniques Used: Amplification, Polymerase Chain Reaction, In Situ Hybridization, Staining, Hybridization

14) Product Images from "A Novel Combination of Factors, Termed SPIE, which Promotes Dopaminergic Neuron Differentiation from Human Embryonic Stem Cells"

Article Title: A Novel Combination of Factors, Termed SPIE, which Promotes Dopaminergic Neuron Differentiation from Human Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006606

Confirmation of microarray results by RT-PCR. cDNA of PA6-DA, PA6-X and MM55K cell lines were analyzed by RT-PCR using gene specific primers for the five candidate genes selected from the microarray data. RT-PCR analysis reproduced the results of cDNA microarray for all five genes, including SDF-1, IGF2, IGFBP4, PTN, and EFNB1. Amplification was performed for 35 cycles. GAPDH was amplified simultaneously as an internal control under the same conditions.
Figure Legend Snippet: Confirmation of microarray results by RT-PCR. cDNA of PA6-DA, PA6-X and MM55K cell lines were analyzed by RT-PCR using gene specific primers for the five candidate genes selected from the microarray data. RT-PCR analysis reproduced the results of cDNA microarray for all five genes, including SDF-1, IGF2, IGFBP4, PTN, and EFNB1. Amplification was performed for 35 cycles. GAPDH was amplified simultaneously as an internal control under the same conditions.

Techniques Used: Microarray, Reverse Transcription Polymerase Chain Reaction, Amplification

15) Product Images from "Bovine Leukemia Virus DNA in Human Breast Tissue"

Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

Journal: Emerging Infectious Diseases

doi: 10.3201/eid2005.131298

Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
Figure Legend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.
Figure Legend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

16) Product Images from "Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo"

Article Title: Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0040012

Minor Populations of Wild-Type KP9 Virus Can Be Detected by qRT-PCR during Chronic Infection SIV Gag KP9 qRT-PCR analysis was performed on serial RNA samples extracted from plasma from Mane-A*10 pigtail macaques (A) 4292 and (B) 4246 infected with SHIV SF162P3 . Wild-type and escape mutant KP9 viruses are represented by closed squares and open triangles, respectively, throughout. Cloning and sequencing (open squares) was compared to qRT-PCR (closed squares) to establish the proportions of wild-type KP9 virus in animal 4292 (C). A mean of 25 clones were sequenced across KP9 for each time point.
Figure Legend Snippet: Minor Populations of Wild-Type KP9 Virus Can Be Detected by qRT-PCR during Chronic Infection SIV Gag KP9 qRT-PCR analysis was performed on serial RNA samples extracted from plasma from Mane-A*10 pigtail macaques (A) 4292 and (B) 4246 infected with SHIV SF162P3 . Wild-type and escape mutant KP9 viruses are represented by closed squares and open triangles, respectively, throughout. Cloning and sequencing (open squares) was compared to qRT-PCR (closed squares) to establish the proportions of wild-type KP9 virus in animal 4292 (C). A mean of 25 clones were sequenced across KP9 for each time point.

Techniques Used: Quantitative RT-PCR, Infection, Mutagenesis, Clone Assay, Sequencing

In Vivo Reversion of KP9 Escape Mutant Virus The kinetics of reversion at SIV Gag KP9 was assessed by qRT-PCR from serial RNA samples extracted from plasma in 4 Mane-A*10 negative pigtail macaques infected with the EM KP9 SHIV mn229 reversion (11.2% WT KP9 by qRT-PCR).
Figure Legend Snippet: In Vivo Reversion of KP9 Escape Mutant Virus The kinetics of reversion at SIV Gag KP9 was assessed by qRT-PCR from serial RNA samples extracted from plasma in 4 Mane-A*10 negative pigtail macaques infected with the EM KP9 SHIV mn229 reversion (11.2% WT KP9 by qRT-PCR).

Techniques Used: In Vivo, Mutagenesis, Quantitative RT-PCR, Infection

17) Product Images from "Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni"

Article Title: Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni

Journal: eLife

doi: 10.7554/eLife.41337

Quantitative PCR to estimate efficiency of programmed mutagenesis. ( A ) The three primers (tri-primer set): Smω1-OVR-F (probe at gRNA, expected DSB and KI sites as red bar and arrow), Smω1-OUT-F and shared Smω1-reverse primer locations used in SYBR green-based PCR to estimate efficiency of on-target, programmed CRISPR/Cas9 gene editing. Estimation of gene-editing efficiency was determined as described ( Shah et al., 2015 ; Yu et al., 2014 ). ( B ) Relative fold amplification efficiency; the ratio of OVR:OUT (gray bar) was set at 100% for control DNA sample, and little or no reduction was observed in relative fold amplification compared to other control groups, including groups exposed to culture medium only, sgRNA only, Cas9 only, ssODN only, heat-killed pLV-ω1X6 virions and ssODN. By contrast, there was reduced relative fold amplification of 2.5%, 6.9%, 4.5% and 12.5% in the experimental groups, RNP, RNP and ssODN, pLV-ω 1X6 virions, and pLV-ω 1X6 virions and ssODN, respectively. Percentages of relative fold amplification differed significantly among control and experiment groups. Means ± SE were established from eleven biological replicates.
Figure Legend Snippet: Quantitative PCR to estimate efficiency of programmed mutagenesis. ( A ) The three primers (tri-primer set): Smω1-OVR-F (probe at gRNA, expected DSB and KI sites as red bar and arrow), Smω1-OUT-F and shared Smω1-reverse primer locations used in SYBR green-based PCR to estimate efficiency of on-target, programmed CRISPR/Cas9 gene editing. Estimation of gene-editing efficiency was determined as described ( Shah et al., 2015 ; Yu et al., 2014 ). ( B ) Relative fold amplification efficiency; the ratio of OVR:OUT (gray bar) was set at 100% for control DNA sample, and little or no reduction was observed in relative fold amplification compared to other control groups, including groups exposed to culture medium only, sgRNA only, Cas9 only, ssODN only, heat-killed pLV-ω1X6 virions and ssODN. By contrast, there was reduced relative fold amplification of 2.5%, 6.9%, 4.5% and 12.5% in the experimental groups, RNP, RNP and ssODN, pLV-ω 1X6 virions, and pLV-ω 1X6 virions and ssODN, respectively. Percentages of relative fold amplification differed significantly among control and experiment groups. Means ± SE were established from eleven biological replicates.

Techniques Used: Real-time Polymerase Chain Reaction, Mutagenesis, SYBR Green Assay, Polymerase Chain Reaction, CRISPR, Amplification

18) Product Images from "Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street? 1"

Article Title: Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street? 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.115.127969

Quantitative real-time PCR analysis of selected genes for RNA-Seq validation (experiment 2). Across 20 comparisons (10 genes × 2 heifer groups [pregnant vs. cyclic]), the expression pattern of the selected genes obtained by qPCR was consistent with the results from the RNA-Seq analysis in all but two cases ( QRFPR and CCL20 ). For each transcript, bars with different superscripts differ significantly ( P
Figure Legend Snippet: Quantitative real-time PCR analysis of selected genes for RNA-Seq validation (experiment 2). Across 20 comparisons (10 genes × 2 heifer groups [pregnant vs. cyclic]), the expression pattern of the selected genes obtained by qPCR was consistent with the results from the RNA-Seq analysis in all but two cases ( QRFPR and CCL20 ). For each transcript, bars with different superscripts differ significantly ( P

Techniques Used: Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Expressing

19) Product Images from "Intracerebral Recruitment and Maturation of Dendritic Cells in the Onset and Progression of Experimental Autoimmune Encephalomyelitis"

Article Title: Intracerebral Recruitment and Maturation of Dendritic Cells in the Onset and Progression of Experimental Autoimmune Encephalomyelitis

Journal: The American Journal of Pathology

doi:

MIP-3α and CCR6 transcripts are up-regulated in the CNS of SJL mice with PLP 139-151-induced EAE. RNA was extracted from different CNS areas and from control tissues (spleen and small intestine), reverse-transcribed, and subjected to PCR amplification using MIP-3α- and CCR6-specific primers, as described in Materials and Methods. A RT-PCR using glyceraldehyde-3-phosphate dehydrogenase-specific primers is also shown as an internal control. RNA was extracted from control mice (PBS/CFA-injected) and from mice at the indicated EAE stages. The data shown were obtained in mice with grade 3 acute EAE and grade 4 relapsing EAE. The PCR products were visualized by ethidium bromide staining. Lane 1 , cerebrum; lane 2 , cerebellum; lane 3 , spinal cord; lane 4 , spleen; and lane 5 , small intestine.
Figure Legend Snippet: MIP-3α and CCR6 transcripts are up-regulated in the CNS of SJL mice with PLP 139-151-induced EAE. RNA was extracted from different CNS areas and from control tissues (spleen and small intestine), reverse-transcribed, and subjected to PCR amplification using MIP-3α- and CCR6-specific primers, as described in Materials and Methods. A RT-PCR using glyceraldehyde-3-phosphate dehydrogenase-specific primers is also shown as an internal control. RNA was extracted from control mice (PBS/CFA-injected) and from mice at the indicated EAE stages. The data shown were obtained in mice with grade 3 acute EAE and grade 4 relapsing EAE. The PCR products were visualized by ethidium bromide staining. Lane 1 , cerebrum; lane 2 , cerebellum; lane 3 , spinal cord; lane 4 , spleen; and lane 5 , small intestine.

Techniques Used: Mouse Assay, Plasmid Purification, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Injection, Staining

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Polymerase Chain Reaction:

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Article Snippet: .. PCR amplification of the 16 exons was performed using the GoTaq PCR core systems I (Promega, Madison, Wisconsin) or the GC-rich PCR system (exon 3 and 9) (Roche, Hvidovre, Denmark). .. PCR products were purified on a NucleoFast96PCR plate (Machery-Nagel, Düren, Germany), directly sequenced on both strands using BigDyeTerminator V1.1, and subsequently resolved on an ABI3130 (Applied Biosystems, Foster City, USA).

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Article Title: Differential expression of p53, p63 and p73 protein and mRNA for DMBA-induced hamster buccal-pouch squamous-cell carcinomas
Article Snippet: .. The PCR amplification reaction mixture (with a final volume of 100 µl) contained diluted, first-strand cDNA reaction product (20 µl; < 10 ng/µl), cDNA reaction dNTPs (2 µl; 200 µ m each), MgCl2 (4 µl; 2 m m ), ×10 reverse transcription buffer (8 µl; 10 m m Tris–HCl, pH = 9.0, 50 m m KCl, 0.1% Triton® X-100), upstream primer (50 pmol), downstream primer (50 pmol) and Taq DNA polymerase (2.5 units; Promega, catalogue number M7660). .. The PCR steps were carried out on a DNA thermal cycler (TaKaRa MP, Tokyo, Japan).

Article Title: Differentiating mouse embryonic stem cells express markers of human endometrium
Article Snippet: .. The RT product was used in PCR with the GoTaq Core System I (Promega, Fitchburg, WI, USA) for 35 amplification cycles at a 57–60°Celsius (C) annealing temperature. .. A 25 μl reaction contained 2 ul MgCl2 (1 mM), 5 ul 5X Green Flexi Buffer (1X), 0.5 ul PCR nucleotide mix (0.1uM), 1 ul each of forward and reverse primers (0.1uM), 0.125 ul of GoTaq polymerase (0.625 units), 2 ul of cDNA, and nuclease free water to make final volume 25 ul.

Article Title: Culture-independent identification of gut bacteria correlated with the onset of diabetes in a rat model
Article Snippet: .. PCR was performed with the GoTaq PCR core system (Promega, Madison, WI, USA). .. The mixtures contained 5 μl of 10 × PCR buffer, 200 μ m dNTPs, 100 μ m of each primer, 2.5 U of Taq polymerase and approximately 100 ng of DNA template in a final volume of 50 μl.

Article Title: Over-expression of SOCS-3 Gene Promotes IL-10 Production by JEG-3 Trophoblast Cells ☆
Article Snippet: .. First-strand cDNA (2 µl) was then used as a template for PCR by GoTaq PCR Core System I (Promaga, Madison, MI). ..

Article Title: Acute effect of infection by adipogenic human adenovirus Ad36
Article Snippet: .. PCR core system II (cat # M7665, Promega) was used for the amplification of DNA. .. Negative PCR controls were water and DNA from uninfected rats.

Article Title: Cytostatic Factor Proteins Are Present in Male Meiotic Cells and ?-Nerve Growth Factor Increases Mos Levels in Rat Late Spermatocytes
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Amplification:

Article Title: Identification of Six Novel PTH1R Mutations in Families with a History of Primary Failure of Tooth Eruption
Article Snippet: .. PCR amplification of the 16 exons was performed using the GoTaq PCR core systems I (Promega, Madison, Wisconsin) or the GC-rich PCR system (exon 3 and 9) (Roche, Hvidovre, Denmark). .. PCR products were purified on a NucleoFast96PCR plate (Machery-Nagel, Düren, Germany), directly sequenced on both strands using BigDyeTerminator V1.1, and subsequently resolved on an ABI3130 (Applied Biosystems, Foster City, USA).

Article Title: Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1? and Tumor Necrosis Factor ?
Article Snippet: .. PCR products were amplified using GoTaq PCR Core System I (M7660, Promega) and analyzed by 2% agarose gel electrophoresis. ..

Article Title: Differential expression of p53, p63 and p73 protein and mRNA for DMBA-induced hamster buccal-pouch squamous-cell carcinomas
Article Snippet: .. The PCR amplification reaction mixture (with a final volume of 100 µl) contained diluted, first-strand cDNA reaction product (20 µl; < 10 ng/µl), cDNA reaction dNTPs (2 µl; 200 µ m each), MgCl2 (4 µl; 2 m m ), ×10 reverse transcription buffer (8 µl; 10 m m Tris–HCl, pH = 9.0, 50 m m KCl, 0.1% Triton® X-100), upstream primer (50 pmol), downstream primer (50 pmol) and Taq DNA polymerase (2.5 units; Promega, catalogue number M7660). .. The PCR steps were carried out on a DNA thermal cycler (TaKaRa MP, Tokyo, Japan).

Article Title: Differentiating mouse embryonic stem cells express markers of human endometrium
Article Snippet: .. The RT product was used in PCR with the GoTaq Core System I (Promega, Fitchburg, WI, USA) for 35 amplification cycles at a 57–60°Celsius (C) annealing temperature. .. A 25 μl reaction contained 2 ul MgCl2 (1 mM), 5 ul 5X Green Flexi Buffer (1X), 0.5 ul PCR nucleotide mix (0.1uM), 1 ul each of forward and reverse primers (0.1uM), 0.125 ul of GoTaq polymerase (0.625 units), 2 ul of cDNA, and nuclease free water to make final volume 25 ul.

Article Title: Acute effect of infection by adipogenic human adenovirus Ad36
Article Snippet: .. PCR core system II (cat # M7665, Promega) was used for the amplification of DNA. .. Negative PCR controls were water and DNA from uninfected rats.

Agarose Gel Electrophoresis:

Article Title: Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1? and Tumor Necrosis Factor ?
Article Snippet: .. PCR products were amplified using GoTaq PCR Core System I (M7660, Promega) and analyzed by 2% agarose gel electrophoresis. ..

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    Promega pcr mix
    Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase <t>PCR,</t> using primers from 5 BLV genome regions, was used to amplify products from <t>DNA</t> extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
    Pcr Mix, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega pcr master mix
    Amplification pattern by <t>RT-PCR</t> with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers <t>inF-F</t> and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.
    Pcr Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

    Journal: Emerging Infectious Diseases

    Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

    doi: 10.3201/eid2005.131298

    Figure Lengend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

    Article Snippet: Control testing was run simultaneously with each batch of human tissue assays: 1) positive control, a smear of BLV-positive cells (FLK cell line) reacted with complete PCR mix; 2) negative controls, a smear of FLK cells and an adjacent serial section of each specimen reacted with PCR mix minus primers, to rule out false-positive reactions unique to each tissue resulting from unquenched endogenous peroxidase, nonspecific reaction of the sheep antibodies used in the final immunoperoxidase detection, or nonspecific DNA repair by Taq polymerase; 3) permeabilization control for entry of PCR mix into cells, with an adjacent serial section of each tissue reacted with PCR mix different from that for IS-PCR by omission of primers, 4.5 mmol/L concentration for MgCl2 , and use of a different Taq polymerase (Promega) that reacts nonspecifically at cooler temperatures (4°C–50°C) to repair DNA.

    Techniques: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

    Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

    Journal: Emerging Infectious Diseases

    Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

    doi: 10.3201/eid2005.131298

    Figure Lengend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

    Article Snippet: Control testing was run simultaneously with each batch of human tissue assays: 1) positive control, a smear of BLV-positive cells (FLK cell line) reacted with complete PCR mix; 2) negative controls, a smear of FLK cells and an adjacent serial section of each specimen reacted with PCR mix minus primers, to rule out false-positive reactions unique to each tissue resulting from unquenched endogenous peroxidase, nonspecific reaction of the sheep antibodies used in the final immunoperoxidase detection, or nonspecific DNA repair by Taq polymerase; 3) permeabilization control for entry of PCR mix into cells, with an adjacent serial section of each tissue reacted with PCR mix different from that for IS-PCR by omission of primers, 4.5 mmol/L concentration for MgCl2 , and use of a different Taq polymerase (Promega) that reacts nonspecifically at cooler temperatures (4°C–50°C) to repair DNA.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

    Expression of modB and mrpB in response to As(III) exposure. (A) RT-PCR cDNA amplified from wild-type strain 5A RNA. Lane assignments: lane 1, molecular mass standards; sizes in kilobases are noted to the left of image; lane 2, early-log-phase, As(III)-naïve cells; lane 3, As(III)-exposed cells; lanes 4 and 5, amplicons derived from RT-PCRs using primers specific for the 16S rRNA as internal controls and corresponding to lanes 2 and 3, respectively. (B) mrpB :: lacZ reporter gene activity recorded with MSUAt2 (closed symbols) and wild-type 5A (open symbols). Reporter enzyme units are Δ A 415 · min −1 · culture optical density (measured as A 595 ) −1 . Cultures were incubated without As(III) (squares) or with 100 μM As(III) throughout the entire incubation (triangles) or spiked with 100 μM As(III) at mid-log phase (circles). Error bars represent 1 standard error of the mean calculated from triplicate cultures. Some standard errors are hidden by the symbols.

    Journal: Journal of Bacteriology

    Article Title: A Na+:H+ Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

    doi: 10.1128/JB.188.4.1577-1584.2006

    Figure Lengend Snippet: Expression of modB and mrpB in response to As(III) exposure. (A) RT-PCR cDNA amplified from wild-type strain 5A RNA. Lane assignments: lane 1, molecular mass standards; sizes in kilobases are noted to the left of image; lane 2, early-log-phase, As(III)-naïve cells; lane 3, As(III)-exposed cells; lanes 4 and 5, amplicons derived from RT-PCRs using primers specific for the 16S rRNA as internal controls and corresponding to lanes 2 and 3, respectively. (B) mrpB :: lacZ reporter gene activity recorded with MSUAt2 (closed symbols) and wild-type 5A (open symbols). Reporter enzyme units are Δ A 415 · min −1 · culture optical density (measured as A 595 ) −1 . Cultures were incubated without As(III) (squares) or with 100 μM As(III) throughout the entire incubation (triangles) or spiked with 100 μM As(III) at mid-log phase (circles). Error bars represent 1 standard error of the mean calculated from triplicate cultures. Some standard errors are hidden by the symbols.

    Article Snippet: DNA was verified to be absent by PCRs with PCR mixtures containing 50 ng of RNA preparation, Tfl DNA polymerase (Promega), and 0.4 μM of each primer of the modB gene (forward primer, 5′-CTTGTGTATAAGAGTCAGCCC-3′; reverse primer, 5′-GACGATTGTGGGATTATGGCT-3′).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Activity Assay, Incubation

    Cartoon illustration characterizing the Tn 5 -B22 insertions in each mutant and the genomic DNA PCR cloned from A. tumefacien s strain C58 for complementation experiments. For each mutant, the gray arrows represent specific genes and their orientation as they occur in the A. tumefaciens C58 genome and as they were PCR cloned into their respective mutants. Small horizontal bars underneath the gray arrows indicate regions of the respective mutant genome that were sequenced via primer walking experiments. (A) Tn 5 -B22 insertion in an mrpB homologue (annotated as mnh in strain C58). Point of insertion was estimated to be at nt 8 based on nucleotide alignments with the C58 strain mrpB / mnhB . (B) Tn 5 -B22 insertion that approximately bisects the modB gene (based on alignment with the A. tumefaciens C58 modB ). Point of insertion was estimated to be at nt 299 based on nucleotide alignments with the C58 strain homologue. Tn 5 -B22 insertion points are indicated by the inverted arrowheads. Positions of the priming sites (identified in Results) used for amplification are shown.

    Journal: Journal of Bacteriology

    Article Title: A Na+:H+ Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

    doi: 10.1128/JB.188.4.1577-1584.2006

    Figure Lengend Snippet: Cartoon illustration characterizing the Tn 5 -B22 insertions in each mutant and the genomic DNA PCR cloned from A. tumefacien s strain C58 for complementation experiments. For each mutant, the gray arrows represent specific genes and their orientation as they occur in the A. tumefaciens C58 genome and as they were PCR cloned into their respective mutants. Small horizontal bars underneath the gray arrows indicate regions of the respective mutant genome that were sequenced via primer walking experiments. (A) Tn 5 -B22 insertion in an mrpB homologue (annotated as mnh in strain C58). Point of insertion was estimated to be at nt 8 based on nucleotide alignments with the C58 strain mrpB / mnhB . (B) Tn 5 -B22 insertion that approximately bisects the modB gene (based on alignment with the A. tumefaciens C58 modB ). Point of insertion was estimated to be at nt 299 based on nucleotide alignments with the C58 strain homologue. Tn 5 -B22 insertion points are indicated by the inverted arrowheads. Positions of the priming sites (identified in Results) used for amplification are shown.

    Article Snippet: DNA was verified to be absent by PCRs with PCR mixtures containing 50 ng of RNA preparation, Tfl DNA polymerase (Promega), and 0.4 μM of each primer of the modB gene (forward primer, 5′-CTTGTGTATAAGAGTCAGCCC-3′; reverse primer, 5′-GACGATTGTGGGATTATGGCT-3′).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Clone Assay, Chromosome Walking, Amplification

    Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.

    Journal: BMC Microbiology

    Article Title: Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

    doi: 10.1186/1471-2180-11-94

    Figure Lengend Snippet: Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.

    Article Snippet: PCR was performed individually using PCR Master Mix and the primer pair inF-F and inF-R for intron-F and inG-F and inG-R for intron-G which we newly designed.

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control

    Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).

    Journal: Journal of Bacteriology

    Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense

    doi: 10.1128/JB.187.7.2416-2425.2005

    Figure Lengend Snippet: Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).

    Article Snippet: The obtained cDNA was amplified by using PCR master mix (Promega) and primer pairs mmpF1 plus mmpB1 and mmpF2 plus mmpB2 , which amplify 309- and 367-bp fragments of the mms16 gene, respectively.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction