Thymus dna extraction Purpose




Дата канвертавання27.04.2016
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Thymus DNA Extraction
Purpose:

In this lab, you are going to extract the DNA from thymus gland nuclei. The thymus is a gland that is very large in immature mammals. It serves a function as a part of the immune system so there are many white blood cells with many large nuclei. Many thousands of thymus cells will be used for the extraction, so you will be combining the DNA from thousands of nuclei. In this way, you should be able to see long, combined strands of DNA.


Materials for 16 lab stations:

Test tubes, 16 Test tube racks, 16 Clinical centrifuge

Prep buffer NaCl Solution EDTA Solution

SDS Solution Ice cold 95% ethanol Small cups or beakers for solutions

Thymus Blender 15 ml cap-less centrifuge tubes, 8

Hand Strainer 250 ml beakers Graduated cylinder, for measuring

Glass stir rods Freezer Refrigerator

Plastic bulb pipets or 100 – 1000 l Pipetmen & blue tips


Procedure:

  1. Isolate nuclei. this step will be done earlier, or as a demonstration

    1. Puree 10 g thymus gland with ~10 ml prep buffer in blender

    2. Mix pureed thymus with 200 ml prep buffer

    3. Stir until well blended.

    4. Strain to remove large chunks of tissue – save liquid & toss solids.
      a – d can be done up to 24 hours ahead of time. Store in refrigerator.

    5. Centrifuge 4 ml of strained liquid for 5 minutes. This makes a pellet of the nuclei. (Nuclei are the heaviest part of the cell.)

    6. Pour off supernatant. (The liquid on top of the pellet.)

    7. Resuspend pellet in 2 ml (~44 drops) prep buffer. Mix well with plastic pipet. Make sure all of pellet is dissolved.

    8. Put ½ of solution into each of 2 test tubes, ~ 1 ml per tube. Resuspended pellet is shared by two lab groups.

    9. OPTIONAL – Prepare a slide with a drop of this solution; stain with methylene blue; observe with a microscope; diagram and describe what you see. This may be unsuccessful if the thymus has been frozen.

  2. Lyse nuclei.

    1. Add ½ ml (~11 drops) of EDTA solution (This binds Mg and Ca ions which are needed by enzymes lurking in the cytoplasm from degrading the DNA as it is released from the nuclei.) Mix gently.

    2. Add 100 l (~2 drops) of SDS solution. (A biological detergent similar to shampoo which solubilizes proteins and disorients fats in the cell membranes.) Mix gently.

    3. Add 250 l (~5 drops) of NaCl solution, one drop at a time, mixing gently after each drop.

  3. Precipitate DNA

    1. Gently add ~1 ml about ½ dropperfull) of ice cold 95% ethanol by pipetting slowly down the side of the test tube. The alcohol will form an overlay. You should begin to see strings of DNA precipitating at the point of the overlay and reaching up into the ethanol layer. (DNA precipitates because it is not soluble in ethanol. All of the other components of the cell are soluble in the ethanol.)

    2. Gently spool DNA threads where the ethanol and DNA mixture meet. There should be gobs of DNA. It is white, stringy and looks a bit like mucus. (Thus the name “Snot on a Stick”.)



Student Procedure for DNA Extraction



    1. Pour off supernatant. (The liquid on top of the pellet.)

    2. Resuspend (remix) pellet in 2 ml (~44 drops) prep buffer. Mix well with plastic pipet. Make sure all of pellet is dissolved – no lumps. Resuspended pellet is shared by two lab groups.

    3. Divide the resuspension evenly into 2 test tubes ( ~ 1 ml per tube)

    4. OPTIONAL – Prepare a slide with a drop of this solution; stain with methylene blue; observe with a microscope; diagram and describe what you see. This may be unsuccessful if the thymus has been frozen.
      Lyse nuclei

    5. Add ½ ml (~11 drops) EDTA solution. Mix gently and let stand 5 minutes.

    6. Add 100 l (~ 2 drops) SDS solutions. Mix gently.

    7. Add 250 l (~ 5 drops) NaCl solutions, one drop at a time, mixing gently after each drop.
      Precipitate DNA

    8. Gently add ~1 ml (about ½ droppers full) of ice cold 95% ethanol by pipetting slowly down the side of the test tube. The alcohol should form an overlay, not mixing with the bottom layer. You should begin to see strings of DNA precipitating at the point of the overlay and reaching up into the ethanol layer. (DNA precipitates because it is not soluble in ethanol. All of the other components of the cell are soluble in the ethanol.)

    9. Gently spool the by putting the stirring rod in just to the interface. Gently twist and spin the rod. Do not stir. The DNA threads will wind onto the rod. There should be gobs of DNA.

    10. Slowly pull the rod out of the tube and observe the DNA. The rod may have to be run up the side of the tube to keep the DNA on it. DNA is white, stringy and looks a bit like mucus. (Thus the name “Snot on a Stick”.)



Analysis:
1. What is the purpose of centrifuging the thymus mixture after it has been pureed and strained?

2. Why do you need to bind Mg and Ca ions?

3. What common household chemicals contain SDS?

4. Why is a salt solution added?

5. What properties of DNA are responsible for its precipitation in alcohol?

6. If you looked at the spooled material under the microscope, would you be able to see the nucleotides that are the building blocks of DNA?

7. Why might freezing the thymus alter the ability to see nuclear material in the cells (step 4)?

8. Compare the general structure of an animal cell with a plant cell and a bacterial cell:


a. what additional step would you have to take to extract the DNA of plant or bacterial cells?

b. What could you use to accomplish that process?




Teacher’s Directions
This lab has been field tested and is appropriate for any level biology class by altering discussion questions. Preparation is fairly simple once the timing for thymus prep and centrifugation is determined. Working solutions are easy to prepare and can be stored indefinitely. Student procedures are not highly specific and are forgiving of inevitable errors. Solutions are not dangerous provided common sense safety precautions are used: do not ingest solutions or place near eyes or mouth. There should be a high yield of DNA, however, it is very crude and most likely could not be used for further electrophoresis. The lab can be performed in one class period with sufficient time for prelab and post lab discussion. Also, a copy of Watson and Crick’s paper describing the “Molecular Structure of Nucleic Acids” (Nature, April 25, 1953, Vol 171, No. 4356, pp. 737-738). It is very readable and provides a good background for discussion. Prelab explanations provided a good opportunity to review cell structure, cell membrane, components, and cell transport mechanisms and how/why they pertain to the procedure. Post lab discussion introduces general recombinant DNA principles (i.e. electrophoresis) and bioethics.
This Lab is designed for 3 sections of 16 extractions.
Working Solutions:

Preparation Buffer Sodium Dodecyl Sulfate (aka sodium laurel sulfate)

57.0 g Sucrose 25 g SDS

3.1 g MgCl2-6H2O distilled H2O to a final volume of 250 ml

0.6 g Tris.HCl Caution – this is a detergent, bubbles easily

400 ml distilled H2O Store at room temperature

Adjust to pH 7.5 w/ 0.1 N NaOH

Bring to a final volume of 500 ml Sodium Chloride

Store in refrigerator. 29.2 g NaCl

Long term storage should be in freezer. Distilled H2O to a final volume of 250 ml

Store at room temperature



Ethylene diamine tetra acetic acid

0.72 g EDTA

200 ml distilled H2O Denatured 95% Ethanol

Bring to a final volume of 250 ml Adjust to pH 7.5 w/ 0.1 N NaOH



Store at room temperature Store in freezer (use ice cold)
Answers to Analysis:

  1. Centrifugation concentrates the heavy cellular material, which will include the nuclei.

  2. DNases, enzymes produced by organisms to break down DNA, need magnesium and calcium ions to “work”.

  3. Detergent used in shampoo and to clean dishes. It disrupts lipids in cell membranes.

  4. Salt will “cover” the acid charge (because of the phosphates) of DNA making the molecule “neutral” so it can precipitate in ethanol.

  5. See #4

  6. You cannot see individual DNA nucleotides because they are too small. You will see long aggregate strands of DNA and associated proteins. This is an excellent time to discuss DNA packaging with histones as well as indi9cate that the next step is to “clean-up” the DNA. This can be accomplished in a few ways: phenol and chloroform, which is too hazardous for school use or DNA extraction kits. Please note, most of the kits need a vacuum line which is not often available in a classroom.

  7. The cells freeze, the expanding water breaks cellular membranes, including nuclear membranes.

  8. a. Eliminate cell walls.
    b. Enzymes, chloroform will break plant cell walls.


Hints


  1. The proportion of thymus to prep buffer needs to be kept the same as in the original recipe. (10 g thymus into 200 ml prep buffer)

  2. The proportions of extract solution to SDS, NaCl. EDTA and ethanol need to be the same as in the original recipe.

  3. It is a good idea to add the prep buffer to the ground up thymus slowly. If the pink color begins to disappear, do not add the full amount.

  4. When too little DNA is available, you must observe quickly. You may be able to see white threads floating up into the ethanol, but they disappear quickly, within 10 – 20 seconds.

  5. The tip of the spooling rod should not go below the interface of the ethanol and the extract solution.

  6. Make sure the thymus is being ground by the blender, sometimes the small chunks slide under the blades, rather than through them. Your solution should be pinkish.

  7. After sitting for a few minutes, a whitish solute should be seen dropping to the bottom of the beaker. These are your nuclei.

  8. If you do not have a centrifuge, you can let the solution sit for an hour or so (or overnight) and the nuclei will drop to the bottom of the beaker. You then pour off the top part of the solution. Keep refrigerated.


Troubleshooting



Problem

Why

Solution

Little to no DNA in tube

DNA is too dilute

Try spinning 8 ml of thymus in prep buffer instead of 4 ml.

Use more thymus or less buffer.

Stir the thymus solution before taking the 4 ml. (Because the nuclei drop, the top part of the solution becomes nuclei deficient.)


Not spooling

DNA not sticking to rod

Try a plastic pipet, coffee stirrer, or roughen up the sides of the glass rod.

Swirl the rod around gently, and then run it up the side of the tube.

Make sure that the tip of the spooling rod does not go into the aqueous phase.


Calf thymus solution is not pink

DNA is too dilute

Grind up some more thymus and put into same buffer. Do not use any new buffer.

Do not need so much solution

Doing fewer experiments

Use half as much thymus and half as much buffer. Do the rest of the experiment the same.

Ethanol does not float on top

Ethanol solution is too dilute

Use a higher percentage alcohol.

Can’t spin solution

No centrifuge

Make the thymus solution a day early and let sit in the refrigerator overnight. Carefully remove beaker from refrigerator and pour off the top part. Do not pour out any of the solute.

Can’t weigh the thymus

No balance

10 g of thymus should be a little bigger than the size of your thumb.

Add the prep buffer to the thymus solution slowly. If the pink starts to disappear, stop adding. Do not add more than the recipe says.




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