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Input material (e.g., same number of cells) such that the variation in the reference gene is < 1 x CT unit. During type I normalization, only CT values of a single primer pair are compared with each other. Hence amplification efficiency differences between primer pairs do not enter the calculation. In contrast, type II normalization compares two different primers pairs, such as for gene A and gene GAPDH, with associated, possibly different, amplification efficiencies kA and kGAPDH. After both...

Analysis of Real Time QPCR and Real Time QPCR Arrays

Theoretical Considerations Prior to reaching saturation (owing to exhaustion of primers and nucleotides, loss of polymerase activity, and so on), PCR amplification proceeds exponentially and can be described by Ni N0 x (1 + k)i, where N0 represents the number of molecules in the original sample and Ni the number of mRNA molecules at cycle i (i 1-40). During the exponential phase, the amplification efficiency k (0 < k < 1) of a given primer pair is constant. Before real-time PCR, it...

Recombinant Technology Approach

Verma, Ke Lan, and Erle S. Robertson Herpesvirus genetics have long been hindered by the large size of the typical herpesvirus genome and the consequent recalcitrance of these genomes to manipulation by standard molecular genetics techniques. However, two primary strategies have emerged that allow for the generation of targeted viral mutants. With these mutants, investigators can pursue critical questions regarding the relationship between specific viral genetic...

Cluster Analysis

Cluster analysis is an unsupervised statistical technique that examines the interpoint distances between all the samples and represents that information in the form of a 2D plot, known as a dendrogram (32). Such dendrograms present the data from high-dimensional row spaces in a form that facilitates the use of human pattern recognition abilities. To generate the dendrogram, cluster analysis methods form clusters of samples based on their nearness in row space. A common approach is to treat...

Construction of Mutants by Recombination Using Linearized Targeting Vectors

Construction of the Targeting Vector 3.1.1.1. Targeting Vectors for Recombination With recA In many cases, the construction of the targeting vector makes use of standard recombinant DNA technology that is widely available (1). We therefore restrict our discussion to some general remarks. Gene deletion is performed by exchanging the gene of interest against an antibiotic resistance cassette (e.g., against kanamycin or tetracyclin). In favorable cases, the gene of interest will contain...

Ntc

Cell E6 E7 S9 E6 copy E7 copy line Ct Quantity6 Ct Quantity Ct Quantity no.c no.c C33A 31.12 1.50E + 02 31.34 1.40E + 01 22.13 3.70E + 01 4.05E + 00 3.78E-01 CaSki 21.02 1.40E + 05 21.07 4.20E +04 26.15 3.40E-03 4.12E + 07 1.24E + 07 HeLa 30.72 2.00E + 02 31.15 1.60E + 01 22.08 2.10E-01 9.52E + 02 7.62E + 01 SiHa 20.48 2.10E + 05 20.67 5.80E + 04 23.72 4.00E-02 5.25E + 06 1.45E + 06 6 Value obtained after extrapolating Ct to quantity axis in absolute standard curve. c Value obtained after...

Herpes Life Cycle Exam Short Notes

The application of modern methods in molecular biology and biotechnology to the study of human, animal, and plant viruses continues to revitalize the age-old discipline of virology. Modern virology remains at the vanguard of contemporary biomedical research largely owing to the impact of viruses in human disease and pathogenesis, but also because of the utility of viruses as model systems for investigation of basic biological processes. DNA Viruses Methods and Protocols describes innovative...