polymerase chain reaction pcr mixture  (PerkinElmer)

Journal: Journal of Clinical Microbiology

Article Title: Use of PCR with Universal Primers and Restriction Endonuclease Digestions for Detection and Identification of Common Bacterial Pathogens in Cerebrospinal Fluid

doi:

Figure Lengend Snippet: Determination of the sensitivity of the universal PCR. Serial 10-fold dilutions of E. coli and S. aureus samples were amplified with the universal PCR, and the PCR products were electrophoresed on an agarose gel. (A) PCR results with E. coli DNA from 10 8 to

Article Snippet: A reaction mixture containing approximately 50 ng of template DNA, PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl2 ; 0.001% gelatin), a 0.2 μM concentration of each PCR primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 2.5 U of Taq DNA polymerase (Perkin-Elmer, Norwalk, Conn.) in a total volume of 50 μl was prepared.

Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Journal: Journal of Virology

Article Title: Array Analysis of Viral Gene Transcription during Lytic Infection of Cells in Tissue Culture with Varicella-Zoster Virus

doi: 10.1128/JVI.77.21.11718-11732.2003

Figure Lengend Snippet: RT-PCR analysis of VZV intergenic DNA. Control (C) and VZV-infected (V) cell RNA was incubated with (+) or without (−) reverse transcriptase and PCR amplified with selected intergenic (Int)-specific primers. Amplified products were compared with those after PCR amplification of VZV DNA. The predicted size of the amplification product (amplicon size) for each Int region primer set is listed. Int regions 6 to 8 map between ORFs 60 and 61; Int regions 10 to 12 map between ORFs 61 and 62 and contain the IR L /IR S junction; and Int regions 1 to 5 map between ORFs 62 and 63 and contain the VZV DNA origin replication (ori).

Article Snippet: Each 100-μl reaction mixture consisted of 0.3 μg of plasmid DNA or 106 VZV DNA molecules in 1× PCR buffer, 1.2 pmol of each primer, 1.5 μmol of MgCl2 and 0.5 to 1.0 units of Taq (PerkinElmer Life Sciences, Boston, Mass.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Incubation, Polymerase Chain Reaction, Amplification

Journal: Journal of Clinical Microbiology

Article Title: Development and Clinical Evaluation of a Highly Sensitive DNA Microarray for Detection and Genotyping of Human Papillomaviruses

doi: 10.1128/JCM.42.7.3272-3280.2004

Figure Lengend Snippet: Microarray design for HPV genotyping. (A) Eight-well platform hybridization reaction chamber. Each well contains 84 probes corresponding to the PCR control, the HPV-positive control, the HPV-negative control, and type-specific probes. (B) Schematic diagram of the HPV DNA microarray probe positions. GAPDH cDNA, HPV-positive controls (P.C), and HPV-negative controls (N.C) were spotted on the center of the slide. Each HPV type-specific probe was printed on the both sides, as shown, and all probes were printed in duplicate.

Article Snippet: To avoid contamination of the template with DNA from a previous PCR, we pretreated the PCR mixture with uracil N ′-glycosylase (0.5 U; Perkin-Elmer) at 50°C for 5 min before PCR ( ).

Techniques: Microarray, Hybridization, Polymerase Chain Reaction, Positive Control, Negative Control

Journal: Journal of Clinical Microbiology

Article Title: Development and Clinical Evaluation of a Highly Sensitive DNA Microarray for Detection and Genotyping of Human Papillomaviruses

doi: 10.1128/JCM.42.7.3272-3280.2004

Figure Lengend Snippet: Detection limit and reproducibility of the HPV DNA microarray. (A) Agarose gel electrophoresis of the MYH-PCR products with a serial dilution of the plasmid with the HPV-16-specific probe. Ten microliters of each 50-μl PCR product was separated on a 1.0% agarose gel. The input copy numbers of the plasmid with the HPV-16-specific probe are as follows in the indicated lanes: M, 1-kb ladder; 1, 10 10 copies; 2, 10 9 copies; 3, 10 8 copies; 4, 10 7 copies; 5, 10 6 copies; 6, 10 5 copies; 7, 10 4 copies; 8, 10 3 copies; 9, 10 2 copies; 10, 10 copies; 11, 1 copy. (B) HPV DNA microarray hybridization results with the amplicon obtained by MYH-PCR of the plasmid with the HPV-16-specific probe. Ten microliters of the 50-μl PCR products derived from 10 5 to 10 0 copies were hybridized with HPV DNA on the microarray for 2 h at 55°C. The starting plasmid copy number is shown at the bottom. (C) Standard curves of signal intensities for HPV-16 with serial dilutions of 10 6 to 10 0 copies. Linear regression was based on the titration series for the plasmid with the HPV-16-specific probe, and each curve is based on the average of three replicates. (D) Reproducibility of the HPV DNA microarray. Three independent experiments were performed with the genomic DNA of Caski cells. The mean ± standard deviation was calculated from the signal intensity of each probe at 635 nm. Each probe is indicated at the bottom: GAPDH, PCR control; Positive, HPV-positive control; HPV-16, HPV-16-specific probe.

Article Snippet: To avoid contamination of the template with DNA from a previous PCR, we pretreated the PCR mixture with uracil N ′-glycosylase (0.5 U; Perkin-Elmer) at 50°C for 5 min before PCR ( ).

Techniques: Microarray, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Hybridization, Amplification, Derivative Assay, Titration, Standard Deviation, Positive Control

Journal: Journal of Virology

Article Title: Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway

doi: 10.1128/JVI.75.6.2857-2865.2001

Figure Lengend Snippet: Activity of viruses with Tcf sites in the E2 promoter. (A) Western blot showing DBP expression 24 h after infection of SW480, H1299, and WI38 with the indicated viruses. (B) E2 and E3 exon structure showing the position of the RT-PCR primers and Taqman probes. (C) PCR quantitation of adenoviral E2 and E3 mRNA by Taqman assay (upper two panels) and adenoviral genomic DNA by Sybr green assay (lower panel) 24 h after infection of SW480 and H1299 with the indicated viruses. (D) Burst assay for virus production by SW480, H1299, and WI38 48 h after infection with the indicated viruses. The viral titer was measured by plaque assay on SW480, which is permissive for all the viruses.

Article Snippet: TaqMan PCRs were performed using a TaqMan Universal PCR Master Mix kit (Perkin-Elmer), a 900 nM concentration of primers (Microsynth and Eurogentec), and 500 nM TaqMan probe (Eurogentec).

Techniques: Activity Assay, Western Blot, Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitation Assay, TaqMan Assay, SYBR Green Assay, Plaque Assay

Journal: Journal of Virology

Article Title: Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway

doi: 10.1128/JVI.75.6.2857-2865.2001

Figure Lengend Snippet: Activity of viruses with Tcf sites in the E1B and E2 promoters. (A) Western blot showing DBP and E1B 55K expression 24 h after infection of SW480, H1299, and WI38 with the indicated viruses. (B) E1B exon structure showing the position of the RT-PCR primers and Taqman probe. (C) PCR quantitation of adenoviral E1B and E2 mRNA (upper two panels) and adenoviral genomic DNA (lower panel) 24 h after infection of SW480 and H1299 with the indicated viruses. (D) Burst assay for virus production by SW480, H1299, and WI38 48 h after infection with the indicated viruses. The viral titer was measured by plaque assay on SW480.

Article Snippet: TaqMan PCRs were performed using a TaqMan Universal PCR Master Mix kit (Perkin-Elmer), a 900 nM concentration of primers (Microsynth and Eurogentec), and 500 nM TaqMan probe (Eurogentec).

Techniques: Activity Assay, Western Blot, Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitation Assay, Plaque Assay