BIOTECH Project: Restriction Digests of DNA

University of Arizona

Name:

Date:

Manipulating DNA and Genetic Engineering: Restriction Digests of DNA

How can we take genes from one organism and connect them with genes from another organism?

Why would we want to do this?

Restriction digest plan

You will be digesting your DNA with two different enzymes. In one tube you will use HindIII and the other EcoRI.

Step 1:Label your tubes with your initials and the enzyme you’re using (H for HindIII and E for EcoRI).

Step 2:Pipette 10 µl water into the bottom of each tube.

Please check off each component as you add it to your reaction tube. You can add these in any order.

Lambda DNADone

Add 6 microliters (µl) of DNA to each tube.

Buffer (Why do we add buffer?)Done

Add 2 microliters (µl) of buffer to your EcoRI tube.

Add 2 microliters (µl) of buffer to your HindIII tube.

EnzymeDone

Add 2 microliters (µl) of the EcoRI enzyme to your EcoRI reaction tube.

Add 2 microliters (µl) of the HindIII enzyme to your HindIII reaction tube.

Why do you only add 2 µl of buffer?

When you’ve visited all the stations, put your tube into the 37°C heat block or water bath. Why do we put the reactions at 37°C?

Analyzing Your Restriction Digest Data

Materials

DNA from restriction enzyme digests (BE SURE TO ADD 5 MICROLITERS LOADING DYE!!)

agarose

Tris-acetate/EDTA solution (TAE)

micropipette/tips

electrophoresis apparatus

Procedure:

1. Get your electrophoresis apparatus. Make sure the comb is in place (there should be one comb near the black electrode) and that there are stoppers at both ends of the gel space.

2. Pour hot agarose into the gel space until it reaches the top of the gel box. Let the agarose harden, which should take about 10 minutes. Don’t touch/move your gel until it’s hard. Why not?

3. While the agarose is hardening add 5 l of loading dye to each of your samples. What is the purpose of the dye?

4. When the agarose gel is hard, take out the comb and stoppers.

5. Load your RESTRICTION DIGEST SAMPLES WITH LOADING DYE into the wells near the BLACK ELECTRODE. Why near the black electrode? Be sure to keep track of which samples you loaded in which lanes.

Draw a picture of your gel and indicate which samples are in which wells before you add DNA to the gel.

6. After you have added your samples pour TAE solution over your gel so that is it completely covered plus a little more. What do you think the TAE solution is for?

7. Run that gel!! Plug the electrodes into your gel box (red to red, black to black), being careful not to bump your gel too much. Plug the power source set between 100 and 125 V into an outlet. How can you tell your gel is running?

After about 30 minutes the DNA should be sufficiently separated to analyze, the purple dye will have migrated approximately 2/3 of the gel, turn off the power and carefully remove the gel. The gel is very fragile, take care to not break it. You can remove the tray that you poured agarose on to and gently slide the gel into the staining tray.

At this point you cannot see the DNA, what can you see and how do the four different lanes compare? Draw a picture of what you see.

Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel with methylene blue and cover try with saran wrap. Stain overnight.

Next day--Viewing the gel: Discard the methylene and carefully place the gel onto a white light box. The gel is very fragile so take care to not break it. Draw a picture of your stained gel, be as accurate as possible in drawing the bands:

How many cuts did each restriction enzyme make?

Analysis of Restriction Digests of DNA:

Table 1

Hind III

Actual Base Pairing (Bp) Sequence / Measured Distance (mm)
23,130
9,416
6,557
4,361
2,322
2,027
564 (may not be detected)
125 (may not be detected)

Based on the DNA fragments you see with the HindII cut, how many nucleotides is the uncut DNA? Does this confirm with the literature on lambda DNA?

Graph your results for HindIII digest to determine the sizes of the EcoRI digest. Do those fragments add up to the size of Lambda DNA? If not, provide possible explanation(s) as to why not.

Table 2

EcoRI

Measured Distance (mm) / Interpolated Bp Sequence / Actual Bp Sequence
Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Band 7
Band 8

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