Isolation of Dna from Hog or Calf Thymus

Experiment No. 5

ISOLATION OF DNA FROM HOG OR CALF THYMUS

Methodology

Materials:

0.15 M NaCl-0.014 M sodium citrate buffer pH 7.4

2.0 M NaCl 95% ethanol

70% ethanol 0.10 M acetate buffer pH 4.6

Diphenylamine reagent Hog or calf thymus

5% trichloroacetic acid (TCA)

0.1% DNA solution

Equipments:

Top loading balance Homogenizer or blender

Centrifuge Thermometer

Micropipettes and pipette tips

1 mL quartz cuvette Stirring rod

UV spectrophotometer

Procedure

A. Extraction of DNA

1. Remove residual fat, blood vessels, and other extraneous tissues from hog or calf thymus.

2. Weigh 12 g of thymus and mince finely.

3. Add 0.15 M NaCl-0.01 M sodium citrate buffer, pH 7.4 to a final volume of 30 mL. Homogenize

at high speed for 10 minutes.

4. Centrifuge the homogenate at 6000 rpm for 10 minutes and discard the supernatant.

5. Suspend the residue in 24 mL 2.0 M NaCl and using the J-tube stir vigorously for 1 minute.

6. Centrifuge for 10 minutes at 6000 rpm. Transfer the supernatant to a beaker and discard the

residue.

7. To the supernatant, slowly add an equal volume of cold 95% ethanol along the sides of the

beaker to form two layers. A fibrous precipitate should form at the interface. Carefully stir the

solution using a pre-weighed stirring rod.

8. Wash the DNA fibers with cold 70% ethanol.

9. Weigh the stirring rod with the DNA fibers and calculate the percent yield.

10. Suspend the DNA fibers in 12 mL 0.15M NaCl-Na citrate buffer pH 7.4.

11. Store sample in the refrigerator until the next meeting.

B. Spectrophotometric Determination of DNA Concentration

1. To 1.5 mL of the tissue extract, add