DNA Isolation and Restriction Enzyme Analysis

In this experiment you will perform DNA isolation under two experimental conditions:one using a buffer containing the detergent SDS and one without detergent. The alternate hypothesis for this experiment might be that the sample prepared without SDS, a strong anionic detergent that breaks apart cell membranes, will yield less DNA than the sample prepared with SDS. The null hypothesis for this experiment might be that both samples will yield an equal amount of DNA.

To begin collect a tissue sample for DNA isolation. Here you will be using your own cheek cells. Use a Popsicle stick to scrape the insides of your cheeks for approximately 30 seconds.

Position a funnel in a 15-milliliter conical tube. Then take 10 milliliters of the 1%saline solution provided to you in a labeled cup and swish it around in your mouth without swallowing. Continue swishing for 90 seconds and then spit the saline solution containing your cheek cells into the funnel to collect in the conical tube.

Next label two empty microcentrifuge tubes with your initials. Label one of the tubes as plus SDS and the other as minus SDS to differentiate the two treatments. Then add 1.5 to two milliliters of the saline solution containing the cheek cells to each tube.

Spin the tubes in a microcentrifuge for 30 seconds. Remove the tubes from the microcentrifuge and carefully pour off the supernatant without disturbing the cells collected at the bottom of each tube. Fill the tubes with more of the saline solution containing the cheek cells and repeat the centrifugation.

Repeat this process taking care to evenly distribute cells between the SDS-positive and SDS-negative tubes until all of the cheek cells have been collected. Next add one milliliter of lysis buffer containing SDS to the tube marked as plus SDS, and then resuspend the cell pellet by pipetting up and down. Then add one milliliter of lysis buffer without SDS to the tube marked with minus SDS, and resuspend the cell pellet by pipetting up and down.

After this add 10 microliters of proteinase K to each tube and then carefully place the tubes in the 56 degrees Celsius water bath for 10 minutes. After 10 minutes carefully remove the tubes from the water bath and add 100 microliters of 2.5 molar sodium chloride to each tube. Close the tubes firmly and invert several times to mix.

Next, label one 15-milliliter conical tube as plus SDS and another with minus SDS. Then pour the contents of each microcentrifuge tube into the corresponding 15-milliliter conical tube. Obtain one 10-milliliter aliquot of 100%ethanol from the freezer.

Hold one of the conical tubes at an angle and slowly dispense one milliliter of 100%ethanol into the tube to form a layer on top of the liquid already in the tube. Repeat this process for the second conical tube. Now look at both of your tubes.

There should be a stringy precipitate present at the interface of the ethanol and the aqueous layer below it. This stringy material contains the isolated DNA. Take pictures of the DNA in the tube and make clear descriptive notes and drawings of your observations in your notebook.

Then using a glass stir rod pick up the DNA from the SDS-positive tube and examine it. Repeat this for the SDS-negative tube, again recording your observations. Now without looking at the labels observe both of your samples.

Do the tubes appear to contain the same amount of precipitated DNA? Does one tube have more precipitated DNA than the other? Using what you have learned about DNA extraction and SDS, can you determine which sample was prepared with SDS and which sample was prepared without SDS?

Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab. To study how restriction enzymes work first navigate to the website listed on the screen. The resources available at this site allow researchers to run virtual experiments before they are performed in the lab.

Click on Tools and Resources to find the NEBcutter tool. Open the program. Then to the right of the search box find the list that says Viral phage.

From that list select Lambda. This is the name of the DNA you will be digesting. Select the Linear option where the tool says, The sequence is and the NEB enzymes option where the tool says, Enzymes to use.

Then click Submit. The next page will display a full restriction map of the lambda DNA. This restriction map can be used to predict what will happen if certain enzymes are used.

Next, click on Custom digest under the Main options heading. Search for the enzyme Stu1 and note that it works optimally in buffer 2.1 or CS buffer. Select the Stu1 enzyme and click digest.

In the next window click on View gel under the Main options header. Select the option to run a virtual 1%gel using a pBR322-BstN1 digest. After the results appear take a screenshot and paste it into a blank document.

After this, navigate back to the main page of the NEBcutter tool and select New custom digest. This time search for the BsrG1 enzyme and note that it performs optimally in buffer 2.1 or buffer 3.1. Select the enzyme and click digest.

Under the Main options header click on View gel and select the options to run a virtual 1%gel using a pBR322-BstN1 digest. Take a screenshot of the results and save this in a text document. Finally, repeat these steps using both Stu1 and BsrG1 together in a single digest.

Again, take a screenshot and save the resulting gel. Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, Stu1 enzyme, and BsrG1 enzyme into an ice bucket, and take this to your work area. The tube labeled Stu1 one will contain the Stu1 digest.

The tube labeled BsrG1 will contain the BsrG1 digest. And the tube labeled S plus B will contain the digest with both of the enzymes. You will now use the appropriate micropipetters to set up the reactions listed in this table.

First, pipette 40 microliters of water into each tube. Then using a fresh pipette tip each time dispense five microliters of buffer 2.1 into each tube and pipette up and down to mix. Next, add four microliters of lambda DNA to each tube, and then pipette 0.5 microliters of each enzyme into the appropriate tubes.

Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 microliters. Next, incubate the tubes at 37 degrees Celsius for 15 to 60 minutes in a water bath and return the buffers and enzymes to the freezer. To prepare an agarose gel to run the digest in a 250-milliliter flask mix one gram of agarose powder with 100 milliliters of TAE buffer.

Microwave the solution for approximately 90 seconds at full power taking care to prevent the solution from boiling over. Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 minutes. While the agarose solution cools set up a casting tray.

When the agarose solution is cool add an appropriate amount of 10, 000X SYBR Safe gel stain to the solution. Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus. Pour the agarose gel solution into the casting tray and allow it to solidify.

Then pull the comb straight up to remove it from the gel. And remove the dams from the casting tray. Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer enough to completely cover the gel.

Remove your samples from the water bath. Then add 8.3 microliters of 6X loading dye to each sample. Dispense 10 microliters of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.

Then load 25 microliters of sample Stu1 into the second well. Place 25 microliters of sample B into the third well and finally load 25 microliters of sample SB into the fourth well. To run the gel plug the leads into the electrophoresis chamber and then into the power supply.

The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive. Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.

After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank. Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator. Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.

Compare the gel you ran in the lab to your virtual enzymatic digest with Stu1. The virtual digest with Stu1 resulted in seven bands. Count the bands in the Stu1 lane of your gel.

Did your gel confirm this prediction? Now look at the results for your BsrG1 digest. The virtual digest with BsrG1 resulted in six bands.

Count the bands in the BsrG1 lane. Did your gel confirm this prediction? Finally, look at the virtual digest with Stu1 and BsrG1.

The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel. Did your gel confirm this prediction?

Does the DNA banding pattern look similar to the virtual digest?