基因工程chapter_2.pptVIP

Context DNA isolation RNA isolation Electrophoresis Southern blot Northern blot Western blot PCR Mutagenesis Transformation Sequencing O ligo synthesis DNA labeling DNA isolation RNA ISOLATION Electrophoresis The Theory of Electrophoresis PAGE electrophoresis coating is the yin and yang polarization, the role imposed on the voltage, with the charge to the mobile ion coating cathode, And with the cathode surface have not formed the basic Dissolved, deposition in the workpiece surface. The SDS gel Equipment The result The agarose gel The result Overview of southern blot Theoretical Basis of SouthernHybridization and Washing Theoretical Basis of Southern Hybridization and Washing Washing: to removes non-specifically bound probe molecules. Variables that affect stringency of washes include: salt concentration, temperature, and SDS concentration Theoretical Basis of Chemiluminescent Detection Blocking: performed with BSA to prevent non-specific binding of antibody Antibody Wash: antibody binds to DIG portion of DIG-dUTP incorporated during amplification of PEPCK gene probes Chemiluminescent Detection: phosphatase enzyme conjugated to anti-DIG antibody reacts with substrate emitting photons of light when phosphate is removed Flow Diagram of Chemiluminescent Detection with CSPD Reaction Solution Time Washing 2X SSC, 0.1% SDS 10 min Washing 0.5X SSC, 0.1% SDS 30 min Blocking 0.1 M Malate, 0.15 M NaCl,1% Blocking Reagent 30 min Antibody Blocking Reagent, 150 mU/ml Anti-Dig Ab 30 min Washing 0.1 M Malate, 0.15 M NaCl, 0.3% Tween 20 30 min Detection CSPD: 0.1 M Tris, 0.1 M NaCl (1:100) 5 min Enhance 37 C incubation 15 min Document ChemiDoc XRS Detection Blot incubated with DIG probe Wash to eliminate non-specifically bound probe molecules Probe detected via DIG specific antibody conjugated to alkaline phosphatase enzyme Phosphatase reacts with substrate emitting photons of light that can be detected via chemidoc system No