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Orientation Lab Safety and Restriction Enzymes. Kabi Neupane, Ph.D. Leeward Community College. Objectives. Familiarize with laboratory safety Learn about Restriction enzymes Perform a restriction digestion of Arabidopsis genomic DNA. Laboratory Safety. Emergency procedures

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orientation lab safety and restriction enzymes

Orientation Lab Safety and Restriction Enzymes

Kabi Neupane, Ph.D.

Leeward Community College

ABE Workshop June 13, 2006

objectives
Objectives
  • Familiarize with laboratory safety
  • Learn about Restriction enzymes
  • Perform a restriction digestion of Arabidopsis genomic DNA

ABE Workshop June 13, 2006

laboratory safety
Laboratory Safety
  • Emergency procedures
  • Eye wash stations
    • Locate both eye wash stations
  • Personal safety
    • Lab coats, gloves, goggles
  • Chemical safety
  • Material Safety Datasheets (MSDS)
    • Red binder located on the back
  • Biological safety

ABE Workshop June 13, 2006

enzymes
Enzymes
  • Enzymes are proteins
    • biological catalysts  help drive biochemical reactions
  • Enzyme names end with an ase(eg., endonuclease)
  • Bacteria have evolved a class of enzymes that destroy foreign DNA (eg. Virus DNA).
    • protect bacteria from bacteriophages (Viruses).
  • Bacteriophages cannot multiply if their DNA is destroyed by the host.

ABE Workshop June 13, 2006

restriction endonucleases
Restriction Endonucleases
  • Restriction endonucleases RESTRICT viruses
    • Viral genome is destroyed upon entry
  • Restriction endonuclease = Restriction enzymes
    • Endo (inside), nuclease (cuts nucleic acid)
  • Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside.
  • The specific DNA sequence is called recognition sequence

ABE Workshop June 13, 2006

restriction enzyme use
Restriction Enzyme Use
  • Discovery of enzymes that cut and paste DNA make genetic engineering possible.
  • Restriction enzyme cuts DNA and generates fragments
  • Ligase joins different DNA fragments
  • DNA fragments from different species can be ligated (joined) to create Recombinant DNA

ABE Workshop June 13, 2006

sticky end cutters

5’ P -

- OH 3’

HindIII

- P 5’

3’ OH -

EcoRI

Sticky End Cutters
  • Most restriction enzymes make staggered cuts
  • Staggered cuts produce single stranded “sticky-ends”
  • DNA from different sources can be spliced easily because of sticky-end overhangs.

ABE Workshop June 13, 2006

blunt end cutters

AluI

HaeIII

Blunt End Cutters
  • Some restriction enzymes cut DNA at opposite base
  • They leave blunt ended DNA fragments
  • These are called blunt end cutters

ABE Workshop June 13, 2006

recognition sequences
Recognition Sequences
  • Many restriction sequences are palindromic. For example,

(Read the same in the opposite direction (eg. madam, race car…)

  • Each restriction enzyme always cuts at the same recognition sequence.
  • 5’ GAATTC 3’
  • 3’ CTTAAG 5’

ABE Workshop June 13, 2006

protection of self dna
Protection of Self DNA
  • Bacteria protect their self DNA from restriction digestion by methylation of its recognition site.
  • Methylation is adding a methyl group (CH3) to DNA.
  • Restriction enzymes are classified based on cognition sequence and methylation pattern.

ABE Workshop June 13, 2006

classification
Classification
  • Type I and III:
    • Large enzyme complex
    • Recognition site is away from the site where the DNA is cleaved
    • Methylation and restriction done by the same enzyme
  • Type IV:
    • Only methylated DNA is cleaved
  • Type II:
    • Recognition and cleavage site are same or close.
    • Restriction and methylation enzymes are different
    • Exclusively used in laboratories

ABE Workshop June 13, 2006

how restriction enzymes are named
How restriction enzymes are named?

ABE Workshop June 13, 2006

cloning vectors
Cloning Vectors

ABE Workshop June 13, 2006

typical restriction digest
Typical Restriction Digest

Sterile, deionized water 16.3 µl

RE 10X Buffer 2.0 µl

Acetylated BSA, 10µg/µl 0.2 µl

DNA, 1µg/µl 1.0 µl

Mix by pipetting, then add:

Restriction Enzyme, 10u/µl 0.5 µl

Final volume 20.0 µl

ABE Workshop June 13, 2006

how does it look after restriction digestion
How does it Look after Restriction Digestion?

Plasmid DNA Digest Genomic DNA Digest

ABE Workshop June 13, 2006