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Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants by Southern Blot Technology (CAT#: STEM-MHT-0127-LGZ)

Introduction

With regard to the commercial application of genetic transformation in woody plants, a major unanswered question is the stability of transgene expression in the same individual over decades. The expression of genes is strongly influenced by the number of copies that have been integrated into the plant genome and the local DNA features near the integration site. The evaluation of the transgene copy number and the identification of the integration site of transgenic woody plants can be realized by using Southern blotting technology.




Principle

Under certain conditions, two single strands of nucleic acid with certain homology can be specifically hybridized to form double strands according to the principle of base complementarity. Generally, DNA molecules to be detected are digested with restriction enzymes, separated by agar-gel electrophoresis, denatured and transferred to nitrocellulocellulose film or nylon film or other solid phase support according to their position in the gel, fixed and then reacted with DNA probes labeled with isotopes or other markers. This is followed by autoradiography or an enzyme reaction to detect the amount of specific DNA molecules. If the object to be tested contains a sequence that is complementary to the probe, the two are combined by the principle of base complementarity, and the free probe is washed and detected by self-development or other suitable techniques, thus revealing the fragment to be tested and its relative size.

Applications

DNA Analysis

Procedure

1. Sample Processing
2. DNA Extraction and Digestion
3. Gel Electrophoresis
4. Gel Pretreatment
5. Transfer membrane
6. Probe Labeling
7. Prehybridization (blocking)
8. Southern hybridization
9. Membrane washing
10. Autoradiographic Assay
11. Results Analysis

Materials

Sample: DNA, Bacterial Fluid/Tissue/Cell
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