Download Lecture 6

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
A Vector
preparation
B Fragment isolation
DNA fragment isolation kit
Lab 7
Ligation of vector and DNA fragment
T4 DNA ligase
How do we ligate a XhoI and
Sal I site together?
GTCGAC
CTGCTG
SalI
CTCGAG
GAGCTC
XhoI
Lab 8
A. 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
-Extract RNA
-RT mRNA
-PCR cDNA
Picture of Plants (WT and mutant)
What are the expression levels of
light regulated genes (chlorophyll
a/b binding proteins CHAB and CLAB)
using tubulin as loading control.
CHAB
CLAB
CHAB or
CLAB
TUB
Isolation of RNA using Commercial Trizol Reagent
Trizol:-mono-phasic solution of phenol and guanidine isothiocyanate
-lyses cells and dissolves cell components while maintaining integrity of RNA
Liquid N2
Trizol
Homogenize
Phenol
Aq Isopropyl
alcohol
Chloroform
Org Ppt RNA
75%
EtOH
DEPC
H2O 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
inactivates RNAses.
Assignment 1
Group
1-1
1-2
1-3
2-1
2-2
2-3
Gel result
no
no
YES
YES
no
no
Group
2-4
2-5
2-6
3-1
3-2
3-3
Gel result
YES
no
no
YES
no
no
Example sequence
GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGA
AGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACA
GAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTG
TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTC
CGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAA
CCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGAT
AAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGC
CAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGG
AAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATC
AACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCG
TGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATC
CTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAA
ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTG
Step into liquid
Assignment 1
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
Discussion of Article 3
Taken from Hall J. C. Science
Taken from Hall Science