Experiment 3: Basic subcloning

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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

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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.

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Lab 6
A Vector preparation
B Fragment isolation
DNA fragment isolation kit
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Lab 7
Ligation of vector and DNA fragment
T4 DNA ligase
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How do we ligate a XhoI and Sal I site together?

• GTCGAC SalI
• CTGCTG
• CTCGAG XhoI
• GAGCTC

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Lab 8
A. Run ligation reactions on an agarose gel
B. Transform bacteria
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Lab 9
Blue white selection (screen)
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Lab 10 and 11

• Small scale DNA isolation (Lab 2)
• Restriction enzyme digestion (Lab 3)

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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.

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Etiolation and det1
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Experimental set up
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• 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 of light regulated
genes (chlorophyll a/b binding proteins CHAB and
CLAB) using tubulin as loading control.
CHAB
CLAB
det1-light
WT-dark
det1-dark
det1-light
det1-dark
WT-light
WT-dark
Ladder
WT-light
-Extract RNA -RT mRNA -PCR cDNA
CHAB or CLAB
TUB
Picture of Plants (WT and mutant)
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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
Phenol Chloroform
75 EtOH
Liquid N2 Trizol
Isopropyl alcohol
Aq
Org
Homogenize
Ppt RNA
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.
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Assignment 1
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Example sequence

• GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCG
  A
• AGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGC
  CACCCGAAAAATACAGTCTTACTGGGCACA
• GAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTA
  TCTGCCAGCGAAGATGCCACTATCAAGGTG
• TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGA
  CAGCGTGCAGGACGTTTCCTTCGACGTCTC
• CGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGG
  ACTTTCACCAGTCATTCGCCTGCGTGAAAA
• CCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAA
  GGGGATTTCGTAGTGAGCGCCTCTAGGGAT
• AAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTT
  AACGGGGCACAGAGAATGGGTACGGATGGC
• CAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAA
  CAGTACGGGTTTGGCACGTGGCAACAAAGG
• AAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCA
  TGGGCACCGGACAGTGCAAGAGCATCGATC
• AACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCT
  CGCATCTGGCTCGCGAGACAAAGTAATTCG
• TGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACG
  ACAACTGGGTTCGCGGCATCGTCTTCCATC
• CTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTA
  TNGGANACGCGCAACANANGGGTAATGAAA
• ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGC
  CATCCTTACGTGGTCNCCGGTAGTG

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Step into liquid
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Assignment 1
I will be available for a large-scale public
consultation on Friday from 400PM 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
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Discussion of Article 3
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Taken from Hall J. C. Science
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Taken from Hall Science
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