Milestones in DNA History

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Introduction of Gene Cloning
Milestones in DNA History 1928 Franklin
Griffith, a British medical officer, discovers
that genetic information can be transferred from
heat-killed bacteria cells to live ones. This
phenomenon, called transformation, provides the
first evidence that the genetic material is a
heat-stable chemical.
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SUMMARYRecombinant DNA technology has enabled
scientists to provide cheap, pure, and readily
available medicines for a variety of illnesses.
By using restriction enzymes, plasmids, and
ligases, we can cut and paste the genes for
almost any protein into bacteria and express
usable proteins. These proteins include human
growth hormone, insulin and many others used to
treat dwarfism, diabetes, stroke, and heart
attacks, as well as cancer.
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• Plasmids
• --- circular DNA molecules, separate from the
  chromosomal DNA.
• --- in bacteria and contain a few non-essential
  genes.
• --- antibiotic resistance.
• --- vectors for gene cloning and are taken into
  cell by transformation

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--- origin of replication (ori) enables them to
replicate within a bacterial cell. --- multiple
restriction enzyme sites --- Reporter Genes and
Proteins antibiotic resistance (ampR) , lacZ
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Restriction enzyme cloning
PCR cloning
--- The new plasmid can be introduced into
bacterial cells that can produce many copies of
the inserted DNA
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• Reference
• www.lakemichigancollege.edu/dept/Arts-Sciences/bio
  /bio111/dna_rna.html
• www.dnalc.org/home.html

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• E. coli strain DH5a competent cell (????)
  for use in heat shock trasformation
• pre-culture in 4-5 ml LB medium in 37? overnight?
• refresh in LB medium by 1100 (100 µl cell in
  final volume 10 ml fresh LB medium for 5
  reactions ) in 37 ?,2 hr (OD value 0.3-0.5)?
• centrifuge with 8000 rpm, 5 min,4??
• cast the supernatant,and resuspend with 100-150
  µl (x5) cold MgCl2 (0.1 M)?
• centrifuge with 5000 rpm,5 min,4??
• cast the supernatant then resuspend with 100-150
  µl (x5) cold CaCl2 (0.1 M)?
• For long term store of competent cells, add
  glycerol to final conc. of 10 and freeze the
  cells on -80 ? freezer.

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• E. coli strain DH5a competent cell for use in
  electroporation
• pre-culture in 4-5 ml LB medium in 37? overnight?
• refresh in LB medium by 1100 (100 µl cell in
  final volume 10 ml fresh LB medium for 5
  reactions ) in 37 ?,2 hr (OD value 0.3-0.5)?
• Chill on ice and centrifuge with 8000 rpm, 5
  min,4??
• cast the supernatant,and resuspend with 5 ml cold
  ice cold distilled water
• centrifuge with 8000 rpm,10 min,4??
• cast the supernatant,and resuspend with 150 µl
  cold ice cold 10 glycerol and spin down
• For long term store of competent cells, add
  glycerol to final conc. of 10 and freeze the
  cells on -80 ? freezer.

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• Heat Shock
• 42 ?, 30 sec
• ???????
• 14 ??
• ?? arial
• ?? ???
• 6/23
• Electroporation
• 12.5 KV / Cm
• 5 msec