Lab 6 Isolation Techniques

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Lab 6Isolation Techniques
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• DNA Isolation from Haman Blood Cells
• Objective
• Isolating DNA molecules from white blood cells
  (WBCs) and visualizing them directly.
• Blood is composed of liquid plasma and solid
  substances including blood cells.
• WBCs have nuclei that contains the nucleic acids.
  
• 1-Cellular membrane and nucleic membrane must be
  destroyed first
• 2- Other components of the cell like proteins
  must be excluded
• 3-finally DNA molecules can be collected and
  directly visualized.

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• In this experiment
• 1- cellular and nucleic membranes are lysed with
  using lysis buffer
• 2- Proteins are precipitated by using phenol and
  chloroform.
• 3- Protect DNA from lysis by DNase enzyme by
  using EDTA which inhibit DNase
• 4- Collect DNA molecules by adding both sodium
  acetate and isopropanol.

Reagents Chemicals such as 1-EDTA (Ethylene
diamine tetra acetate) which removes Mg2 ions
that is essential for preserving the overall
structure of the cell membrane. 2-SDS (Sodium
dodecyl sulfate), which aids in disrupting the
cell membranes by removing the lipids of the cell
membranes, are included in the extraction. 3-buff
er which lysing the cells, the final step in
the preparation of a cell extract is removal of
insoluble cell debris
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• Purification of DNA from cell extract
• In addition to DNA the cell extract will contain
  significant quantities of protein and RNA. A
  variety of procedures can be used to remove these
  contaminants, leaving the DNA in a pure form.
• Reagents
• Phenol
• The standard way to de-proteinize a cell extract
  is to add phenol or a 11 mixture of phenol
  chloroform. These organic solvents precipitate
  proteins but leave the nucleic acids in aqueous
  solutions. The aqueous solution of nucleic acid
  can be removed with a pipette.
• Ribonuclease enzyme
• Ribonuclease enzyme remove RNA by degrade these
  molecules into ribonucleotide subunits.

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• Materials
• - Lysis buffer
• - SE buffer
• Blood with anti-coagulant
• - Proteinase K (10mg/ml)
• - SDS reagent (contains detergent and EDTA)
• - Chloroform/ isoamyl alcohol 241
• - Sodium acetate
• - Isopropanol
• - Micropipettes and tips
• - Refrigerated centrifuge and 15 ml Eppendorf
  tubes

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• Steps
• 1- In Eppendorf tube add 2.5 ml of blood with
  7.5 ml of lysis buffer, shake well, and leave
  it in ice for 5 min.
• 2- Put the sample in the centrifuge (with
  balancing tube of 10 ml water) at 1200 rpm, 4C
  for 10 min.
• 3- Discard supernatant. Add to the pellet 2.5 of
  lysis buffer and centrifuge again at same
  conditions (set balance tube )
• 4- Discard supernatant. Add 1.25 ml SE buffer to
  pellet, centrifuge again at same conditions (set
  balance tube !)
• 5- Discard supernatant. Add 1.25 ml of SE buffer
  10µl proteinase K 60 µl SDS. Shake gently for
  7 min.

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6- Add 1.25 ml SE buffer 2.5 ml phenol. Shake
well for 5 min then centrifuge again at 3000
rpm, 10 C for 5 min. 7- Transfer supernatant
to new tube. Add 2.5 ml Phenol/
chloroform/isoamy alcohol. Shake
well for 5 min, then centrifuge Again at same
conditions. 8- Transfer supernatant to new
tube. Add 2.5 ml chloroform/isoamyl alcohol.
Shake well for 5 min, then centrifuge again at
same conditions. 9- Transfer supernatant to new
tube. Add 75 µl 3M Sodium acetate 2.5 ml
isopropanol. Observe DNA fibers visually.
vortex
shaker
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DNA Isolation from Blood Animation

• http//www.iupui.edu/wellsctr/MMIA/isolating_dna/
  dna_isolation_rev.swf