Lab 8: Extraction of DNA from Wheat Germ
Introduction
The process of DNA extraction from cells is of primary importance to the field of biotechnology and forensics (CSI, anyone? And of course, all forensic scientists are incredibly good-looking, wear sexy clothes while examining dead bodies in ditches, and only work with bright-blue colored liquids ….). It is the first step in the analysis and manipulation of DNA, enabling scientists to detect genetic disorders and to produce DNA fingerprints of individuals. And DNA can, of course, be used to create recombinant organisms for beneficial products, including insulin, antibiotics, and hormones, to name just a few.
In order for DNA to be extracted from plant cells, the cell walls, cell membranes, and nuclear membranes must first be lysed (broken open) and component molecules pulled apart. The addition of hot water, a detergent (sodium dodecyl sulfate, SDS), and a salt solution disrupts the non-covalent associations that hold these membranes together. The DNA can then be released from the nucleus into the solution. Cold ethanol is layered atop the solution, creating two distinct layers. The interface between the water and ethanol allows for precipitation of DNA because DNA is soluble in water and insoluble in ethanol. The precipitated DNA can then be collected and used for further study.
Extraction Procedure
Put on gloves and then:
Lysed:
Weigh out 2 g of raw wheat germ. Record the actual mass. My actual mass was 2.09 grams.
Place the raw wheat germ into a large test tube (25 mm x 150 mm).
Add 10 ml of warm water (50-60 oC) to the test tube, parafilm the top, and invert continuously and gently for one minute. Make sure that you have a tight seal with the parafilm!!
Cut a square of cheese cloth àà four layers
Hold the cheesecloth over the top of a 50 ml beaker and pour the contents of the test tube onto the cheesecloth.
Remove the excess liquid from the wheat germ by gently squeezing the cheesecloth over the beaker. The solution in the beaker will be cloudy and off white.
Rinse out the test tube with distilled water so that there is no residue left inside. Pour the rinse down a sink.
Add the liquid in the beaker back to the test tube.
Add 2 ml of 10% SDS/ 1 .5% NaCl to the test tube.
Parafilm the test tube and mix by inverting the tube back and forth continuously and gently for 30 seconds.
Stand the test tube up in a large beaker (400 or 600 mL size). Use a disposable glass pipette to VERY SLOWLY add 10 mLs cold 100 % ethanol to the tube, one drop at a time, so that the drops run down the side of the tube (see the figure). The alcohol should appear as a distinct layer (approximately 3-5 mm) on top of the wheat germ extract. Do not move the beaker or disturb in any way. DNA precipitation occurs at the aqueousethanol interface. If the ethanol is mixed in rather than gently layered on top of the extract, then the ethanol will become too dilute for the DNA to precipitate out and the results will be much less dramatic.
Let the test tube stand undisturbed for 2 minutes. A white, stringy film should begin to appear at the aqueous ethanol interface. That is DNA.
Insert a glass hook into the test tube putting the bottom end into the white, stringy film. Slowly rotate the glass hook. This process should wrap (or spool) the DNA chains around the bottom of the rod.
After about 30 seconds of ‘spooling’ the DNA, remove the hook from the test tube and closely observe the material sticking to it.
If there still appears to be a film between the phases go ahead and spool some more.
Once you are done spooling, hold the hook inside the top of a test tube and press it against the tube to squeeze out excess liquid. Leave the DNA to air dry by putting it atop of a disposable glass pipette.
Get a disposable test tube and fill it about half way with 75% cold ethanol. Put the loop with DNA into the ethanol and move it up and down a few times to rinse.
Let the DNA air dry for 5 minutes by putting it atop of a disposable glass pipette like before. Record the weight of your loop plus DNA.
Recorded weight of 3.045 grams hook with DNA.
Dissolve the DNA in 10.0 mL of standard saline citrate (SSC, 0.15 M NaCl, .015 M sodium citrate, pH 7.20). It may take awhile for the DNA to fully dissolve. Stir for several minutes with the loop and make sure that there is no material left on the loop. Record the weight of your loop, now that it does not have any DNA attached.
Measure and record the A260 value for your DNA sample. Measure and record the A280 value for your DNA sample. If the values for your A260 and A280 are above 1, you will have to make a 1 to 10 (or more) dilution of your sample in order to bring them down to a value of ≤ 1. Keep track of your dilutions!
Recorded weight of 3.03 grams hook without DNA.
Save your concentrated DNA sample to run on a gel in two weeks. Make sure you label the sample with your names and group. You will be storing it at -20C.
Wheat Germ DNA Extraction Laboratory Report
In the space provide below, draw a four-nucleotide piece (e.g. ATGC) of DNA, drawing out the FULL structure showing the four different nitrogenous bases that occur in DNA and showing it in its ionization state at physiological pH. Draw the four bases (A, C, T, G) and their full structure so the backbone of the DNA and the nitrogenous bases.
Describe the difference between the genomic DNA you isolated in this experiment and “plasmid” DNA used in recombinant DNA technology.
1.6 to 2.0 Ratio = Pure DNA
What is the A260 value for your DNA sample? ____0.334___________ .
What is the A280 value for your DNA sample? ____0.325___________ .
What is the A260/A280 ratio for your DNA sample? _______________ .
Based upon the A260/A280 ratio of your sample, what can you say about the purity of your isolated DNA?
What was the weight of the DNA you isolated? _________________.
You started with 2g of raw wheat germ. Using this knowledge calculate the apparent % of your raw wheat germ that is supposedly DNA. Show your work below.
Apparent % DNA in wheat germ: ____________.
Here is a rule of thumb used to calculate the approximate DNA concentration in a solution, based upon the A260 value: A DNA solution of concentration 50 µg/ml gives an A260 value of 1.
Calculate the total amount of DNA in your isolated wheat germ DNA sample based upon your measured A260. Take into account any dilutions you may have made. Show your calculations below.
Total amount of DNA in wheat germ (based on A260): ____________.
% DNA in wheat germ (based on A260): ____________.
Which method of measuring the amount of DNA in your wheat germ gives the larger amount of DNA?:
Which method do you think is a more accurate way of measuring the amount of DNA in a sample?:Why?
