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DNA Isolation and Restriction Enzyme Analysis

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DNA Isolation and Restriction Enzyme Analysis
 

Procedure

  1. DNA Isolation
    • NOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent SDS and one without detergent. Hypotheses: 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 s. IMPORTANT: Do not scrape hard enough to cause pain or draw blood.
    • Position a funnel in a 15-mL conical tube.
    • Then take 10 mL 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 s 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 +SDS and the other as -SDS to differentiate the two treatments.
    • Then add 1.5 to 2 mL of the saline solution containing the cheek cells to each tube.
    • Spin the tubes in a microcentrifuge for 30 s.
    • 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 and -SDS tubes until all of the cheek cells have been collected.
    • Next, add 1 mL of lysis buffer containing SDS to the tube marked as +SDS, and then resuspend the cell pellet by pipetting up and down.
    • Then add 1 mL of lysis buffer without SDS to the tube marked with -SDS and resuspend the cell pellet by pipetting up and down.
    • After this add 10 µL of proteinase K to each tube and then carefully place the tubes in the 56 ºC water bath for 10 min.
    • After 10 minutes carefully remove the tubes from the water bath and add 100 µL of 2.5 M NaCl to each tube.
    • Close the tubes firmly and invert several times to mix.
    • Next, label one 15 mL conical tube as +SDS and another with -SDS.
    • Pour the contents of each microcentrifuge tube into the corresponding 15 mL conical tube.
    • Obtain one 10 mL aliquot of 100% ethanol from the freezer.
    • Hold one of the conical tubes at an angle and slowly dispense 1 mL 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.
    • Using a glass stir rod, pick up the DNA from the +SDS tube and examine it. Repeat this for the -SDS tube, again recording your observations. Take note of whether one of the tubes appears to have more precipitate than the other, and why this might be.
    • Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab.
  2. Restriction Enzyme Virtual Digest
    • To study how restriction enzymes work, first navigate to the New England Biolabs website: https://www.neb.com/
    • 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 and open the program.
    • 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 StuI and note that it works optimally in buffer 2.1 or CS buffer. Select the StuI 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 BsrGI 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 StuI and BsrGI together in a single digest.
    • Again, take a screenshot and save the resulting gel.
  3. DNA Profiling
    • Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, StuI enzyme, and BsrGI enzyme into an ice bucket, and take this to your work area.
    • Label a tube as StuI – this will contain the StuI digest.
    • Label a tube as BsrGI to contain the BsrGI digest.
    • Then, label a tube S+B, to contain the digest with both of the enzymes.
    • Use the appropriate micropipetters to set up the reactions listed in the Table 1:Click Here to download Table 1
    • First, pipette the 40 µL of water into each tube.
    • Then, using a fresh pipette tip each time, dispense 5 µL of buffer 2.1 into each tube and pipette up and down to mix.
    • Next, add 4 µL of lambda DNA to each tube, and then pipette 0.5 µL 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 µL.
    • Next, incubate the tubes at 37 ºC for 15 - 60 min 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 mL flask mix 1 g of agarose powder with 100 mL of TAE buffer.
    • Microwave the solution for approximately 90 s 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 min.
    • While the agarose solution cools set up a casting tray.
    • When the agarose solution is cool add 5 µL 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.
    • Once the gel is solid, pull the comb straight up to remove it from the gel, and then remove the dams from the casting tray if appropriate.
    • 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 to completely cover the gel.
    • Remove your samples from the water bath.
    • Then add 8.3 µL of 6X loading dye to each sample.
    • Dispense 10 µL of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.
    • Then load 25 µL of sample StuI into the second well.
    • Place 25 µL of sample BsRGI into the third well.
    • Load 25 µL of sample S+B 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. CAUTION: Protect eyes from the UV by wearing goggles or using the provided shield.
    • Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.
  4. Results
    • Compare the gel you ran in the lab to your virtual enzymatic digest with StuI.
    • The virtual digest with StuI resulted in seven bands. Count the bands in the StuI lane of your gel and note whether this matched the virtual digest or appeared to show a similar pattern. NOTE: Small bands on agarose electrophoresis gels are often invisible.
    • Now look at the results for your BsrGI digest. The virtual digest with BsrGI resulted in six bands. Count the bands in the BsrGI lane. Again, note whether the virtual digest and lab digest showed the same or a similar pattern.
    • Finally, look at the virtual digest with StuI and BsrGI. The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel, and consider whether the DNA banding pattern looks similar to the virtual digest.

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