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Extraction and Quantitation of DNA From E. coli. Experiment 6. QUIZ. Write the brief procedure for cloning the gene X from the Vibrio fischeri to the E.coli ? Write the open structure of DNA (3-4 bp ) by using followings? (no need to draw H bonds). S. Pyr. Purine. P.

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Experiment 6

Extraction and Quantitation of DNA From E. coli

Experiment 6


QUIZ

  • Write the brief procedure for cloning the gene X from the Vibriofischerito the E.coli?

  • Write the open structure of DNA (3-4 bp) by using followings? (no need to draw H bonds)

S

Pyr

Purine

P


Why do we isolate DNA ?

  • To perform any experiment with DNA, one should get pure DNA

    • Isolate, analyze, search the genes in different cells, organs, organisms

    • Knock-out the genes and look for their functions

    • Diagnosis of genetic diseases

    • Forensic purposes

    • etc.


Procedure

  • to denature all proteins

  • to get rid of all fatty membranes

  • To dissociate proteins from DNA

  • To inhibit DNAse activity

  • To breakdown membranous structures

  • to provide isotonic medium

-To get rid of growth medium

  • Pour 50 ml of E. coli culture into a 50-ml centrifuge bottle.

  • Centrifuge at 5000 rpm for 5 minutes

  • Discard the supernatant

  • Resuspend the pellet in 6.5 ml of saline-EDTA buffer

  • Centrifuge at 5000 rpm for 5 minutes

  • Pour off the supernatant

  • Add 3.5 ml of saline-EDTA buffer to resuspend the pellet

  • Add 500 µl of SDS

  • Place in a 50 oC water bath for 10 minutes

  • Add 1 ml 5 M NaCl

  • Invert the tube three times to ensure mixing


Procedure, continuing

  • Chloroform causes surface denaturation of proteins, so deproteinizes the DNA solution

  • - Isoamyl alcohol is to reduce foaming, to separate and maintain the stability of the layers of the centrifuged, deproteinized solution.

  • To separate the DNA from the “junk”

  • DNA dissolves in ionic solution, while other macromolecules will not.

  • DNA is precipitated from the ionic solution by ethanol.

  • Add 4 ml of chloroform-isoamyl alcohol (24:1)

  • Shake vigorously for 15 minutes

  • Centrifuge at 5000 rpm for 3 minutes

  • Carefully decant the top layer (this contains the DNA) into 7.5 ml of cool 95% EtOH

  • Taking a glass rod, carefully come down into the viscous material (DNA plus contaminants), noting the spongy texture and the different colored layers. The top layer is milky white and contains the contaminating proteins of the cell.

  • Holding the glass rod, gently stir the solution. Watch the DNA at the bottom of the beaker winding around the stirring rod.


Procedure, continuing

Absorbance at 260 nm

  • Obtain the materials required:

    • TE Buffer

    • Spectrophotometer (with a UV lamp); quartz or silica cuvettes with a 1 cm light path

  • The extract must be diluted into the range of the standard curve. Dilute the extract with TE according to the table bellow. Label the tubes 1 through 3, place 3 ml of the buffer into tube 4 for a blank.


Procedure, continuing

  • Read the absorbance in a spectrophotometer at 260 nm & 280 nm and calculate the average DNA concentration and purity of your sample.

DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit)

1,8 < OD 260 / OD 280 < 2, pure enough

OD 260 / OD 280 < 1,8, high protein rate


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