Lecture 6. Experiment 3: Basic subcloning Flowchart Experiment 4: Light regulated genes Principles and process of RNA isolation Assignment 1 due next week Discussion of Article 3. Experiment 3. Goal: To introduce basic procedures used to manipulate DNA.
B Fragment isolation
DNA fragment isolation kit
Ligation of vector and DNA fragment
T4 DNA ligase
A. Run ligation reactions on an agarose gel
B. Transform bacteria
Blue white selection (screen)
What are the expression levels of
light regulated genes (chlorophyll
a/b binding proteins CHAB and CLAB)
using tubulin as loading control.
Picture of Plants (WT and mutant)
Trizol:-mono-phasic solution of phenol and guanidine isothiocyanate
-lyses cells and dissolves cell components while maintaining integrity of RNA
Chloroform: separate aqueous and organic phases (RNA is in the top aqueous phase)
Isopropanol: Precipitates RNA
75% Ethanol: Wash RNA pellet
DEPC (Diethylpyrocarbonate) water: Resuspend RNA pellet in RNAse-free water
DEPC interacts with the active site histidine residue of RNAse and irreversibly
I will be available for a large-scale public consultation on Friday from 4:00PM until we are done. Bring your laptops.
You can make appointments for consultation as well. Time is running out.
Assignment 1 due next Monday Oct. 27, 2008