Lecture 6 Experiment 3: Basic subcloning Flowchart Experiment 4: Light regulated genes Principles...
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Transcript of Lecture 6 Experiment 3: Basic subcloning Flowchart Experiment 4: Light regulated genes Principles...
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.
• General procedures are DNA fragment isolation, ligation of DNA, transformation of bacteria, small scale DNA isolation.
Lab 6
DNA fragment isolation kit
A Vector preparation
B Fragment isolation
Lab 7
Ligation of vector and DNA fragment
T4 DNA ligase
How do we ligate a XhoI and Sal I site together?
GTCGAC SalI
CTGCTG
CTCGAG XhoI
GAGCTC
Lab 8A. Run ligation reactions on an agarose gel
B. Transform bacteria
Lab 9
Blue white selection (screen)
Lab 10 and 11
• Small scale DNA isolation (Lab 2)
• Restriction enzyme digestion (Lab 3)
Experiment 4
• Goal: to characterize the response of light regulated genes.
• Method: RT-PCR.
• General take home messages: Use of RT-PCR to measure levels of transcript accumulation; use of mutants to characterize a biological process; environmental regulation of gene expression.
Etiolation and det1
Experimental set up
1. wild-type grown in the light
2. wild-type grown in the dark
3. det1 grown in the light
4. det1 grown in the dark
What are the expression levels oflight regulated genes (chlorophylla/b binding proteins CHAB and CLAB)
using tubulin as loading control.
-Extract RNA-RT mRNA-PCR cDNA
WT-
light
WT-
dark
det1
-ligh
tde
t1-d
ark
Ladde
r
WT-
light
WT-
dark
det1
-ligh
tde
t1-d
ark
CHAB CLAB
TUB
CHAB or CLAB
Picture of Plants (WT and mutant)
Isolation of RNA using Commercial Trizol Reagent
Liquid N2
Trizol
Homogenize
PhenolChloroform
RNA
Isopropyl alcohol
Ppt RNA DEPC H2O
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 RNA75% Ethanol: Wash RNA pelletDEPC (Diethylpyrocarbonate) water: Resuspend RNA pellet in RNAse-free water
DEPC interacts with the active site histidine residue of RNAse and irreversibly inactivates RNAses.
Aq
Org
75% EtOH
Assignment 1
Group Gel result Group Gel result1-1 no 2-4 YES1-2 no 2-5 no1-3 YES 2-6 no2-1 YES 3-1 YES2-2 no 3-2 no2-3 no 3-3 no
Example sequence
GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGAAGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACAGAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTG
TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTCCGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAACCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGATAAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGCCAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGGAAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATCAACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCGTGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATCCTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAA
ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTG
Step into liquid
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
Assignment 1
Discussion of Article 3
Taken from Hall J. C. Science
Taken from Hall Science