Isolation of DNA

	THYMUS DNA EXTRACTION David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103 Modified from a protocol by Lana Hays
aliquot taken of blended thymus	This page has been accessed times since 9 January 2003. 30 January 2001, 2 Feb 01, 16 Feb 01	Precipitated DNA can be spooled

The stages of DNA isolation include
    1) homogenization of thymus in a buffered isotonic sucrose-detergent solution
    2) increasing the osmolarity with saline
    3) centrifugation to remove cellular debris
    4) decantation of the supernatant into a clean beaker
    5) precipitation of the DNA by layering cold ethanol on the top of the supernatant
    6) spooling of the precipitated DNA

MATERIALS AND SOLUTIONS

EQUIPMENT

Calf thymus (we purchase it frozen, and it works a second year)
Prep Buffer Solution (0.33 M sucrose, 0.05 M MgSO₄):
    57 g granulated sugar
    1 buffered aspirin
    3 g epsom salts <>Q.s. with dH₂O to 500 mL (pH =~6.7) (turbid)
10% detergent solution (sodium dodecyl sulfate?):
    45 mL distilled water
    5 mL Palmolive liquid detergent
2 M NaCl salt solution:
    11.7 g non-iodized salt Q.s. with dH₂O to 100 ml
100% ethanol, ice cold

knife and cutting board

    balance
    graduated cylinders (10ml and100ml)
    blender
    1 mL & 5 mL displacement pipets and tips
    15 mL centrifuge tubes with caps
    table top clinical centrifuge
    10 or 15 mL beaker
    thin glass rods, hook at end (can be fashioned from pasteur pipet)

PROTOCOL

1. Cut out a chunk of thymus 1 inch square(15 g or so) and place in the clean blender.

2. Measure out:
a) 100 mL of the prep buffer

b) 10 mL of the detergent solution.

3. Add prep buffer and detergent solution to the blenderwith the thymus.
Blend for 1 minute or until the mixture is smooth.

4. Transfer 1mL of the homogenate to a 15 mL centrifuge tube.

If the homogenate stops up the pipet tip, you may snip off 3 mm of the tip to create a larger bore.)

5. Add 2 mL of salt solution to the centrifuge tube, cap, and shake well by hand for 2 minutes.

6. Centrifuge for 7 minutes in a balanced table top centrifuge, top speed (settingof 7) (approximately 50,000 x g).

7. Carefully remove the tube from the centrifuge and note the two phases:
upper layer: supernatant, DNA is dissolved here. To be saved.

lower layer: pellet, cell debris, discard it.

8. Decant the supernatant into a clean 10 mL beaker, avoiding any of the pellet.
Alternatively, especially if purity of DNA is important, the
supernatant should be pipeted off with a wide bore pipet.

(Snip about 1/4 inch off the end of a 1 mL displacement pipet tip.)

9. Carefullyand slowly add 5 mL -20 C 100% ethanol down the side of the beaker to form a layer on top. EtOH is less dense and forms a layer on top of the salty DNA solution.

10. Allow the mixture sit undisturbed for a minuteor two.
(We tried liver (the pinker beakers) and it did not produce significant amounts of DNA in this experiment.)

11. The DNA will precipitate in the alcohol just above the lower aqueous phase and float there as threads. The DNA of the thymus will form long threads that easily spool on a gladd rod.

Here are pictures of students gleefully spooling their DNA in 2005.

These students isolated DNA in 2003:
(Jennifer , Sandra, Kyle, Kelli, Jan, Jan, Mindy.)

Pictures taken 2004:

	1. Cut out a chunk of thymus 1 inch square(15 g or so) and place in the clean blender.
	2. Measure out: a) 100 mL of the prep buffer b) 10 mL of the detergent solution.
	3. Add prep buffer and detergent solution to the blenderwith the thymus. Blend for 1 minute or until the mixture is smooth.
	4. Transfer 1mL of the homogenate to a 15 mL centrifuge tube. If the homogenate stops up the pipet tip, you may snip off 3 mm of the tip to create a larger bore.)
	5. Add 2 mL of salt solution to the centrifuge tube, cap, and shake well by hand for 2 minutes.
	6. Centrifuge for 7 minutes in a balanced table top centrifuge, top speed (settingof 7) (approximately 50,000 x g).
	7. Carefully remove the tube from the centrifuge and note the two phases: upper layer: supernatant, DNA is dissolved here. To be saved. lower layer: pellet, cell debris, discard it.
	8. Decant the supernatant into a clean 10 mL beaker, avoiding any of the pellet. Alternatively, especially if purity of DNA is important, the supernatant should be pipeted off with a wide bore pipet. (Snip about 1/4 inch off the end of a 1 mL displacement pipet tip.)
	9. Carefullyand slowly add 5 mL -20 C 100% ethanol down the side of the beaker to form a layer on top. EtOH is less dense and forms a layer on top of the salty DNA solution.
	10. Allow the mixture sit undisturbed for a minuteor two. (We tried liver (the pinker beakers) and it did not produce significant amounts of DNA in this experiment.)
	11. The DNA will precipitate in the alcohol just above the lower aqueous phase and float there as threads. The DNA of the thymus will form long threads that easily spool on a gladd rod. Here are pictures of students gleefully spooling their DNA in 2005. These students isolated DNA in 2003: (Jennifer , Sandra, Kyle, Kelli, Jan, Jan, Mindy.)

Modified from:

"Generic, All Purpose DNA Extraction from Meat Protocol" Judy Brown

"Mammalian DNA Extraction" Theresa Knapp

Tried mushrooms-they formed a white band at the bottom of theEtOH, just above the aqueous phase. In both cases, forgot to add the detergent...The origianl recipe called for 30 g thymus/ 100 mL buffer, but it formedso much DNA that it glommed up into "snot balls." 30 gm/100 mL of bufferwas probably too much thymus. Should try liver since thymus is hard tofind. (Amy got it frozen in Hyde Park for $10/pound.)

THYMUS DNA EXTRACTION