THYMUS DNA EXTRACTION
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
Modified from a protocol by Lana Hays
This page has been accessed times since 9 January 2003.
30 January 2001, 2 Feb 01, 16 Feb 01
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|
thymus (we purchase it frozen, and it works a
Prep Buffer Solution (500 mL) (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 (100 mL) (sodium dodecyl sulfate?):
45 mL distilled water
5 mL Palmolive liquid detergent
2 M NaCl salt solution (100 mL):
11.7 g non-iodized salt Q.s. with dH2O to 100 ml
100% ethanol, ice cold
knife and cutting board
|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
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.
into a clean 10 mL beaker, avoiding
Alternatively, especially if purity of DNA is important,
(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
Here are pictures of students gleefully spooling their DNA in 2005.
These students isolated DNA in 2003:
(Jennifer , Sandra, Kyle, Kelli, Jan, Jan, Mindy.)
"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.)