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