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Promega pcr cleanup kit
<t>PA3719</t> and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp <t>PCR-generated</t> template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.
Pcr Cleanup Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿"

Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00543-07

PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.
Figure Legend Snippet: PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

Techniques Used: In Vitro, Polymerase Chain Reaction, Generated, Purification, Electrophoresis, Produced

2) Product Images from "Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1"

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

Journal: Journal of Virology

doi: 10.1128/JVI.02444-13

The ribonucleoside 3-deazauridine causes altered mutation spectra and increased mutation frequencies in HIV-1. Targets cells were treated at the EC 75 for each ribonucleoside analog, and at 48 h postinfection, cells were harvested and the total genomic DNA was purified. The HIV-1 pol gene (specifically, the 5′ region of the HIV-1 RT open reading frame, corresponding to amino acids 1 to 366) was amplified by PCR and cloned. (A to G) Tabulated summary of the mutation spectra as a percentage of the total mutations as well as the absolute values. The total number of clones sequenced, the total number of mutations scored, and the total number of bases sequenced are also indicated. Mutation frequency (freq.) was calculated on the basis of the ratio of the total numbers of mutations to the total number of bases sequenced. The fold difference in mutation frequency (Fold Δ mut. freq.) relative to that of the no-drug control was determined.
Figure Legend Snippet: The ribonucleoside 3-deazauridine causes altered mutation spectra and increased mutation frequencies in HIV-1. Targets cells were treated at the EC 75 for each ribonucleoside analog, and at 48 h postinfection, cells were harvested and the total genomic DNA was purified. The HIV-1 pol gene (specifically, the 5′ region of the HIV-1 RT open reading frame, corresponding to amino acids 1 to 366) was amplified by PCR and cloned. (A to G) Tabulated summary of the mutation spectra as a percentage of the total mutations as well as the absolute values. The total number of clones sequenced, the total number of mutations scored, and the total number of bases sequenced are also indicated. Mutation frequency (freq.) was calculated on the basis of the ratio of the total numbers of mutations to the total number of bases sequenced. The fold difference in mutation frequency (Fold Δ mut. freq.) relative to that of the no-drug control was determined.

Techniques Used: Mutagenesis, Purification, Amplification, Polymerase Chain Reaction, Clone Assay

3) Product Images from "Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication"

Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

Journal: BMC Genomics

doi: 10.1186/1471-2164-11-183

Verification of Cct1 and Cct2 roles in modulating FHV replication in infected S2 cells . (A) RT-PCR validation of Cct1 or Cct2 RNAi-mediated knockdown. Drosophila S2 cells were treated with dsRNAs specific for LacZ (lane 1), Cct1 (lane 2), Cct2 (lane 3), or both Cct1 and Cct2 (lane 4) for 72 h, and gene-specific mRNA expression was examined by semi-quantitative RT-PCR as described in Fig. 2, except that only results from cDNA dilutions that produced submaximal signals are shown. (B) Total PC content in cells treated with the dsRNAs described above. PC levels were determined as in Fig. 3. (C) Viability of cells treated with dsRNAs described above determined by MTT assay. (D) FHV RNA accumulation in infected S2 cells after RNAi-mediated knockdown of Cct1 , Cct2 , or both. Mock-infected cells (lane 1), cells treated with the indicated dsRNA as described above or FHV RNA1 as a positive control (lanes 2-6), or treated with 50 μM miltefosine (lane 7), were infected with FHV and viral-specific RNAs or protein were analyzed by northern blotting or immunoblotting 12 h after infection, respectively. For viral RNA analysis, blots for positive-sense (+) and negative-sense (-) genomic RNA1 and subgenomic (+)RNA3 are shown. The decrease in (+)RNA1 accumulation in Cct1 knockdown cells (lane 4) was not consistently seen in all experiments. Ribosomal RNA (rRNA) bands detected by ethidium bromide staining are shown as loading controls. For protein analysis, blots for FHV protein A and the cellular loading control tubulin are shown. (E) Quantitative data for genomic (+)RNA1 and (-)RNA1, subgenomic (+)RNA3, and protein A accumulation in S2 cells treated with the indicated dsRNA or miltefosine after infection with FHV. Results are expressed as percentage of accumulation relative to LacZ dsRNA-treated control. P -value
Figure Legend Snippet: Verification of Cct1 and Cct2 roles in modulating FHV replication in infected S2 cells . (A) RT-PCR validation of Cct1 or Cct2 RNAi-mediated knockdown. Drosophila S2 cells were treated with dsRNAs specific for LacZ (lane 1), Cct1 (lane 2), Cct2 (lane 3), or both Cct1 and Cct2 (lane 4) for 72 h, and gene-specific mRNA expression was examined by semi-quantitative RT-PCR as described in Fig. 2, except that only results from cDNA dilutions that produced submaximal signals are shown. (B) Total PC content in cells treated with the dsRNAs described above. PC levels were determined as in Fig. 3. (C) Viability of cells treated with dsRNAs described above determined by MTT assay. (D) FHV RNA accumulation in infected S2 cells after RNAi-mediated knockdown of Cct1 , Cct2 , or both. Mock-infected cells (lane 1), cells treated with the indicated dsRNA as described above or FHV RNA1 as a positive control (lanes 2-6), or treated with 50 μM miltefosine (lane 7), were infected with FHV and viral-specific RNAs or protein were analyzed by northern blotting or immunoblotting 12 h after infection, respectively. For viral RNA analysis, blots for positive-sense (+) and negative-sense (-) genomic RNA1 and subgenomic (+)RNA3 are shown. The decrease in (+)RNA1 accumulation in Cct1 knockdown cells (lane 4) was not consistently seen in all experiments. Ribosomal RNA (rRNA) bands detected by ethidium bromide staining are shown as loading controls. For protein analysis, blots for FHV protein A and the cellular loading control tubulin are shown. (E) Quantitative data for genomic (+)RNA1 and (-)RNA1, subgenomic (+)RNA3, and protein A accumulation in S2 cells treated with the indicated dsRNA or miltefosine after infection with FHV. Results are expressed as percentage of accumulation relative to LacZ dsRNA-treated control. P -value

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Produced, MTT Assay, Positive Control, Northern Blot, Staining

FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).
Figure Legend Snippet: FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Generated, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Microarray, Produced, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils
Article Snippet: .. PCR products from the primary PCRs were purified by preparative gel electrophoresis (1% low-melting-point NuSieve agar; FMC Bioproducts, Rockland, Maine), followed by purification with the PCR cleanup kit from Promega. ..

Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication
Article Snippet: .. PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction. .. To generate the cDNA templates for Cct1 and Cct2 validation studies, candidate RNAi targets that differed from the O'Farrell library constructs were identified using the E-RNAi program [ ].

Clone Assay:

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1
Article Snippet: .. Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis. .. DNA sequences were aligned and compared to the HIV-1 subtype B NL4-3 sequence using the Seqman program of the Lasergene software package (DNASTAR, Madison, WI).

Ligation:

Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿
Article Snippet: .. Annealing was achieved by incubating 800 pmol of each oligonucleotide in T4 ligation buffer at 95°C for 3 min and allowing the reaction mixture to cool at room temperature for 5 h, after which the 3′ PA3719 DNA was purified using a gel and PCR cleanup kit (Promega). .. PCR-based random mutagenesis ( ) of mexR was carried out using primers RanMuMexRForward (5′-GAGC GGTGACC ATGAACTACCCCGTGAATCC-3′; BstEII site underlined) and RanMuMexRReverse (5′-GAGG CTCGAG TTAAATATCCTCAA-GCGGTTG-C-3′; XhoI site underlined).

Purification:

Article Title: Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils
Article Snippet: .. PCR products from the primary PCRs were purified by preparative gel electrophoresis (1% low-melting-point NuSieve agar; FMC Bioproducts, Rockland, Maine), followed by purification with the PCR cleanup kit from Promega. ..

Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication
Article Snippet: .. PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction. .. To generate the cDNA templates for Cct1 and Cct2 validation studies, candidate RNAi targets that differed from the O'Farrell library constructs were identified using the E-RNAi program [ ].

Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
Article Snippet: .. An identical PCR was then repeated, using 5 μl of the previous reaction as the template, followed by purification with a PCR cleanup kit (Wizard SV, Promega) to remove excess primers. .. Two sequential PCRs were conducted in order to dilute the template relative to the exponentially amplified mutated PCR product, and to compensate for the much higher transformation efficiency of the circular template plasmid compared to the nicked mutant vector (a second, alternative protocol is provided in the Supplementary Data ).

Article Title: Cognitive Decline Is Associated with Reduced Reelin Expression in the Entorhinal Cortex of Aged Rats
Article Snippet: .. PCR products containing T7 and SP6 extensions were purified by SVgel and a PCR cleanup kit (Promega). .. 35S-uridine triphosphate (UTP)–labeled riboprobe was then generated using the Maxiscript kit (Ambion).

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1
Article Snippet: .. Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis. .. DNA sequences were aligned and compared to the HIV-1 subtype B NL4-3 sequence using the Seqman program of the Lasergene software package (DNASTAR, Madison, WI).

Article Title: Effect of Wastewater Treatment Plant Effluent on Microbial Function and Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal Flow
Article Snippet: .. PCRs were purified by using a Promega PCR cleanup kit, ligated into pGEM-T plasmids, and transformed into competent Escherichia coli JM109 cells (Promega, Inc.). .. The DNA sequencing was conducted by the Australian Genome Research Facility (Adelaide) using the M13F primer location.

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase
Article Snippet: .. The PCR products obtained with these oligonucleotides were purified with a PCR cleanup kit (Promega), digested with BamHI and AgeI (Fermentas), and ligated in the plasmid mentioned above. .. Another three different versions of SSAT were produced as follows.

DNA Sequencing:

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1
Article Snippet: .. Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis. .. DNA sequences were aligned and compared to the HIV-1 subtype B NL4-3 sequence using the Seqman program of the Lasergene software package (DNASTAR, Madison, WI).

Polymerase Chain Reaction:

Article Title: Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils
Article Snippet: .. PCR products from the primary PCRs were purified by preparative gel electrophoresis (1% low-melting-point NuSieve agar; FMC Bioproducts, Rockland, Maine), followed by purification with the PCR cleanup kit from Promega. ..

Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication
Article Snippet: .. PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction. .. To generate the cDNA templates for Cct1 and Cct2 validation studies, candidate RNAi targets that differed from the O'Farrell library constructs were identified using the E-RNAi program [ ].

Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
Article Snippet: .. An identical PCR was then repeated, using 5 μl of the previous reaction as the template, followed by purification with a PCR cleanup kit (Wizard SV, Promega) to remove excess primers. .. Two sequential PCRs were conducted in order to dilute the template relative to the exponentially amplified mutated PCR product, and to compensate for the much higher transformation efficiency of the circular template plasmid compared to the nicked mutant vector (a second, alternative protocol is provided in the Supplementary Data ).

Article Title: Cognitive Decline Is Associated with Reduced Reelin Expression in the Entorhinal Cortex of Aged Rats
Article Snippet: .. PCR products containing T7 and SP6 extensions were purified by SVgel and a PCR cleanup kit (Promega). .. 35S-uridine triphosphate (UTP)–labeled riboprobe was then generated using the Maxiscript kit (Ambion).

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1
Article Snippet: .. Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis. .. DNA sequences were aligned and compared to the HIV-1 subtype B NL4-3 sequence using the Seqman program of the Lasergene software package (DNASTAR, Madison, WI).

Article Title: Effect of Wastewater Treatment Plant Effluent on Microbial Function and Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal Flow
Article Snippet: .. PCRs were purified by using a Promega PCR cleanup kit, ligated into pGEM-T plasmids, and transformed into competent Escherichia coli JM109 cells (Promega, Inc.). .. The DNA sequencing was conducted by the Australian Genome Research Facility (Adelaide) using the M13F primer location.

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase
Article Snippet: .. The PCR products obtained with these oligonucleotides were purified with a PCR cleanup kit (Promega), digested with BamHI and AgeI (Fermentas), and ligated in the plasmid mentioned above. .. Another three different versions of SSAT were produced as follows.

Staining:

Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication
Article Snippet: .. PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction. .. To generate the cDNA templates for Cct1 and Cct2 validation studies, candidate RNAi targets that differed from the O'Farrell library constructs were identified using the E-RNAi program [ ].

Transformation Assay:

Article Title: Effect of Wastewater Treatment Plant Effluent on Microbial Function and Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal Flow
Article Snippet: .. PCRs were purified by using a Promega PCR cleanup kit, ligated into pGEM-T plasmids, and transformed into competent Escherichia coli JM109 cells (Promega, Inc.). .. The DNA sequencing was conducted by the Australian Genome Research Facility (Adelaide) using the M13F primer location.

Plasmid Preparation:

Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1
Article Snippet: .. Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis. .. DNA sequences were aligned and compared to the HIV-1 subtype B NL4-3 sequence using the Seqman program of the Lasergene software package (DNASTAR, Madison, WI).

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase
Article Snippet: .. The PCR products obtained with these oligonucleotides were purified with a PCR cleanup kit (Promega), digested with BamHI and AgeI (Fermentas), and ligated in the plasmid mentioned above. .. Another three different versions of SSAT were produced as follows.

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    Promega pcr cleanup kit
    <t>PA3719</t> and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp <t>PCR-generated</t> template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.
    Pcr Cleanup Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dna cleanup kit
    <t>RT-PCR</t> analysis of gene cotranscription in fsr / gelE loci. Every three lanes divided by a solid line represents one set of experiments using the same primers. In each set, the first lane shows PCR using chromosomal <t>DNA</t> as the template, which serves as a positive control for PCR, the second lane shows an RT-PCR with (+) RNA and reverse transcriptase (RTase) but without (−) chromosomal DNA; the third lane shows an RT-PCR with RNA but without RTase and chromosomal DNA, which serves as a control to determine the contamination of DNA in RNA samples. (A to E) RT-PCR analysis using primers covering the intergenic regions between orf1 and fsrA , fsrA and fsrB , fsrB and fsrC , fsrC and gelE , and gelE and sprE , respectively. (F) RT-PCR using two internal primers (lytF1 and lytR1) of E. faecalis autolysin, a positive control for RT-PCR. Primers: OR1, orfRTR1; AF1, fsrARTF1; AR1, fsrARTR1; BF1, fsrBRTF1; BR1, fsrBRTR1; CF1, fsrCRTF1; CR1, fsrCRTR1; EF1, gelERTF1; ER1, gelERTR1; SF1, sprERTF1; LF1, lytF1; LR1, lytR1. (G) Diagram illustrating the positions of the primers used in the RT-PCR analysis. Solid line, chromosomal DNA; boxes, genes; arrows, primers.
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    PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

    Journal: Journal of Bacteriology

    Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿

    doi: 10.1128/JB.00543-07

    Figure Lengend Snippet: PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

    Article Snippet: Annealing was achieved by incubating 800 pmol of each oligonucleotide in T4 ligation buffer at 95°C for 3 min and allowing the reaction mixture to cool at room temperature for 5 h, after which the 3′ PA3719 DNA was purified using a gel and PCR cleanup kit (Promega).

    Techniques: In Vitro, Polymerase Chain Reaction, Generated, Purification, Electrophoresis, Produced

    The ribonucleoside 3-deazauridine causes altered mutation spectra and increased mutation frequencies in HIV-1. Targets cells were treated at the EC 75 for each ribonucleoside analog, and at 48 h postinfection, cells were harvested and the total genomic DNA was purified. The HIV-1 pol gene (specifically, the 5′ region of the HIV-1 RT open reading frame, corresponding to amino acids 1 to 366) was amplified by PCR and cloned. (A to G) Tabulated summary of the mutation spectra as a percentage of the total mutations as well as the absolute values. The total number of clones sequenced, the total number of mutations scored, and the total number of bases sequenced are also indicated. Mutation frequency (freq.) was calculated on the basis of the ratio of the total numbers of mutations to the total number of bases sequenced. The fold difference in mutation frequency (Fold Δ mut. freq.) relative to that of the no-drug control was determined.

    Journal: Journal of Virology

    Article Title: Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

    doi: 10.1128/JVI.02444-13

    Figure Lengend Snippet: The ribonucleoside 3-deazauridine causes altered mutation spectra and increased mutation frequencies in HIV-1. Targets cells were treated at the EC 75 for each ribonucleoside analog, and at 48 h postinfection, cells were harvested and the total genomic DNA was purified. The HIV-1 pol gene (specifically, the 5′ region of the HIV-1 RT open reading frame, corresponding to amino acids 1 to 366) was amplified by PCR and cloned. (A to G) Tabulated summary of the mutation spectra as a percentage of the total mutations as well as the absolute values. The total number of clones sequenced, the total number of mutations scored, and the total number of bases sequenced are also indicated. Mutation frequency (freq.) was calculated on the basis of the ratio of the total numbers of mutations to the total number of bases sequenced. The fold difference in mutation frequency (Fold Δ mut. freq.) relative to that of the no-drug control was determined.

    Article Snippet: Amplicons were resolved on a 0.8% TAE (Tris-acetate-EDTA) DNA gel, purified using a Promega Wizard SV gel and PCR cleanup kit (Madison, WI), and cloned into the pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing analysis.

    Techniques: Mutagenesis, Purification, Amplification, Polymerase Chain Reaction, Clone Assay

    Verification of Cct1 and Cct2 roles in modulating FHV replication in infected S2 cells . (A) RT-PCR validation of Cct1 or Cct2 RNAi-mediated knockdown. Drosophila S2 cells were treated with dsRNAs specific for LacZ (lane 1), Cct1 (lane 2), Cct2 (lane 3), or both Cct1 and Cct2 (lane 4) for 72 h, and gene-specific mRNA expression was examined by semi-quantitative RT-PCR as described in Fig. 2, except that only results from cDNA dilutions that produced submaximal signals are shown. (B) Total PC content in cells treated with the dsRNAs described above. PC levels were determined as in Fig. 3. (C) Viability of cells treated with dsRNAs described above determined by MTT assay. (D) FHV RNA accumulation in infected S2 cells after RNAi-mediated knockdown of Cct1 , Cct2 , or both. Mock-infected cells (lane 1), cells treated with the indicated dsRNA as described above or FHV RNA1 as a positive control (lanes 2-6), or treated with 50 μM miltefosine (lane 7), were infected with FHV and viral-specific RNAs or protein were analyzed by northern blotting or immunoblotting 12 h after infection, respectively. For viral RNA analysis, blots for positive-sense (+) and negative-sense (-) genomic RNA1 and subgenomic (+)RNA3 are shown. The decrease in (+)RNA1 accumulation in Cct1 knockdown cells (lane 4) was not consistently seen in all experiments. Ribosomal RNA (rRNA) bands detected by ethidium bromide staining are shown as loading controls. For protein analysis, blots for FHV protein A and the cellular loading control tubulin are shown. (E) Quantitative data for genomic (+)RNA1 and (-)RNA1, subgenomic (+)RNA3, and protein A accumulation in S2 cells treated with the indicated dsRNA or miltefosine after infection with FHV. Results are expressed as percentage of accumulation relative to LacZ dsRNA-treated control. P -value

    Journal: BMC Genomics

    Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

    doi: 10.1186/1471-2164-11-183

    Figure Lengend Snippet: Verification of Cct1 and Cct2 roles in modulating FHV replication in infected S2 cells . (A) RT-PCR validation of Cct1 or Cct2 RNAi-mediated knockdown. Drosophila S2 cells were treated with dsRNAs specific for LacZ (lane 1), Cct1 (lane 2), Cct2 (lane 3), or both Cct1 and Cct2 (lane 4) for 72 h, and gene-specific mRNA expression was examined by semi-quantitative RT-PCR as described in Fig. 2, except that only results from cDNA dilutions that produced submaximal signals are shown. (B) Total PC content in cells treated with the dsRNAs described above. PC levels were determined as in Fig. 3. (C) Viability of cells treated with dsRNAs described above determined by MTT assay. (D) FHV RNA accumulation in infected S2 cells after RNAi-mediated knockdown of Cct1 , Cct2 , or both. Mock-infected cells (lane 1), cells treated with the indicated dsRNA as described above or FHV RNA1 as a positive control (lanes 2-6), or treated with 50 μM miltefosine (lane 7), were infected with FHV and viral-specific RNAs or protein were analyzed by northern blotting or immunoblotting 12 h after infection, respectively. For viral RNA analysis, blots for positive-sense (+) and negative-sense (-) genomic RNA1 and subgenomic (+)RNA3 are shown. The decrease in (+)RNA1 accumulation in Cct1 knockdown cells (lane 4) was not consistently seen in all experiments. Ribosomal RNA (rRNA) bands detected by ethidium bromide staining are shown as loading controls. For protein analysis, blots for FHV protein A and the cellular loading control tubulin are shown. (E) Quantitative data for genomic (+)RNA1 and (-)RNA1, subgenomic (+)RNA3, and protein A accumulation in S2 cells treated with the indicated dsRNA or miltefosine after infection with FHV. Results are expressed as percentage of accumulation relative to LacZ dsRNA-treated control. P -value

    Article Snippet: PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Produced, MTT Assay, Positive Control, Northern Blot, Staining

    FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).

    Journal: BMC Genomics

    Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

    doi: 10.1186/1471-2164-11-183

    Figure Lengend Snippet: FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).

    Article Snippet: PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction.

    Techniques: Infection, Expressing, Quantitative RT-PCR, Generated, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Microarray, Produced, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    RT-PCR analysis of gene cotranscription in fsr / gelE loci. Every three lanes divided by a solid line represents one set of experiments using the same primers. In each set, the first lane shows PCR using chromosomal DNA as the template, which serves as a positive control for PCR, the second lane shows an RT-PCR with (+) RNA and reverse transcriptase (RTase) but without (−) chromosomal DNA; the third lane shows an RT-PCR with RNA but without RTase and chromosomal DNA, which serves as a control to determine the contamination of DNA in RNA samples. (A to E) RT-PCR analysis using primers covering the intergenic regions between orf1 and fsrA , fsrA and fsrB , fsrB and fsrC , fsrC and gelE , and gelE and sprE , respectively. (F) RT-PCR using two internal primers (lytF1 and lytR1) of E. faecalis autolysin, a positive control for RT-PCR. Primers: OR1, orfRTR1; AF1, fsrARTF1; AR1, fsrARTR1; BF1, fsrBRTF1; BR1, fsrBRTR1; CF1, fsrCRTF1; CR1, fsrCRTR1; EF1, gelERTF1; ER1, gelERTR1; SF1, sprERTF1; LF1, lytF1; LR1, lytR1. (G) Diagram illustrating the positions of the primers used in the RT-PCR analysis. Solid line, chromosomal DNA; boxes, genes; arrows, primers.

    Journal: Journal of Bacteriology

    Article Title: Characterization of fsr, a Regulator Controlling Expression of Gelatinase and Serine Protease in Enterococcus faecalis OG1RF

    doi: 10.1128/JB.183.11.3372-3382.2001

    Figure Lengend Snippet: RT-PCR analysis of gene cotranscription in fsr / gelE loci. Every three lanes divided by a solid line represents one set of experiments using the same primers. In each set, the first lane shows PCR using chromosomal DNA as the template, which serves as a positive control for PCR, the second lane shows an RT-PCR with (+) RNA and reverse transcriptase (RTase) but without (−) chromosomal DNA; the third lane shows an RT-PCR with RNA but without RTase and chromosomal DNA, which serves as a control to determine the contamination of DNA in RNA samples. (A to E) RT-PCR analysis using primers covering the intergenic regions between orf1 and fsrA , fsrA and fsrB , fsrB and fsrC , fsrC and gelE , and gelE and sprE , respectively. (F) RT-PCR using two internal primers (lytF1 and lytR1) of E. faecalis autolysin, a positive control for RT-PCR. Primers: OR1, orfRTR1; AF1, fsrARTF1; AR1, fsrARTR1; BF1, fsrBRTF1; BR1, fsrBRTR1; CF1, fsrCRTF1; CR1, fsrCRTR1; EF1, gelERTF1; ER1, gelERTR1; SF1, sprERTF1; LF1, lytF1; LR1, lytR1. (G) Diagram illustrating the positions of the primers used in the RT-PCR analysis. Solid line, chromosomal DNA; boxes, genes; arrows, primers.

    Article Snippet: The PCR products were purified by a DNA cleanup kit (Promega, Madison, Wis.) and sequenced using the fsrCRTF1 primer.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control