Expt. 2
DNA Restriction Enzyme Digestion and Electrophoresis
Background & Objective:
Analysis of DNA with restriction endonucleases followed by agarose gel electrophoresis of the resulting fragments is one of the most commonly used techniques in molecular biology. Most often the sizes of the resulting unknown DNA fragments are determined by comparison to standard fragments of known size that have been resolved in an adjacent lane. One of the most commonly used of these DNA standards is generated by digesting lambda (λ) DNA with the restriction endonucleases, HindIII. Since you will be running many agarose gels this semester, you will be using HindIII digested λ DNA as an electrophoresis standard in future experiments. The goal of this laboratory is to generate the digested λ standard and confirm its restriction fragment pattern using agarose gel electrophoresis.
Procedures:
Restriction Digestions
1. Label a microfuge tube with λ HindIII.
2. Use the table below to create the reaction mix. Make sure to change pipette tips for each reagent.
λ DNA (0.5 μg/μl)5 μl
10X Restriction Buffer5 μl
HindIII Restriction Enzyme5 μl
Sterile water 35 μl
Total Volume 50 μl
3. Mix the Reaction by vortexing and bring the contents to the bottom of the microfuge tube by briefly centrifuging.
4. Incubate the reaction tube in the 37C water bath for 1 hour.
5. Label an microfuge tube as – Control
6. Use the table below to create the Negative control reaction mix.
λ DNA (0.5 μg/μl)1 μl
10X Restriction Buffer1 μl
Sterile water 8 μl
Total Volume 10 μl
7. Mix the Negative Reaction by vortexing and bring the contents to the bottom of the microfuge tube by briefly centrifuging.
8. Incubate the reaction tube in the 37C water bath for 1 hour.
Pouring an Agarose Gel (1%)
1. Seal the ends of the gel-casting tray with the rubber seals and place it in the electrophoresis unit to create a seal.
2. Dilute 50 mls of 10X TBE with deionized, distilled water to 1X TBE.
3. Weigh 0.5 grams of molecular grade agarose and suspend in 50 mls of 1 TBE in a 300 ml Erlenmeyer flask.
4. Microwave the agarose solution on high power for 1 minute. CAREFULLY, mix the solution, keeping the flask opening pointed away from anyone. Microwave two more times at 15 seconds, carefully mixing in between. The agarose should be completely melted.
5. When the agarose has cooled to below 60C, add 3 μl of ethidium bromide (10 mg/ml) to the agarose solution and gently swirl.
6. Pour the agarose into the casting tray and insert the well-forming comb.
7. When the gel has solidified, about 20-30 minutes, remove the comb, turn the gel tray 90 degrees, and fill the electrophoresis chamber with 1X TBE buffer until just above the gel.
Gel Electrophoresis
1. Remove the restriction digestions from the 37C water bath and centrifuge briefly.
2. Remove 10 μl from the λ HindIII reaction tube and place it in another microfuge tube. Place the remaining 40 μl of the reaction aside.
3. Add 3 μl of loading dye to each the aliquot of the digestion reaction and the negative control reaction. Mix both tubes and centrifuge briefly.
4. Load the wells of the gel as shown in the table below.
Lane 1Positive control given to you by the instructor
Lane 212 μl of the λ HindIII digestion reaction
Lane 312 μl of theNegative control reaction
5. Electrophorese the gel at 110-120V for 1 hour.
6. Photograph the gel on a UV transilluminator using the Kodak software.
7. Compare the size of theλ HindIII digestion reaction to the Positive control
8. If the restriction pattern of your λ HindIII restriction digestion matches the Positive control, then label 5 microfuge tubes with your initials and λ HindIII. Aliquot 8 λ μl into each tube along with 2 μl of loading dye and place in the freezer for future use as size standards.
9.Print a copy of your gel for your notebook.
23,130 base pairs (bp)
9,416 bp
6,557 bp
4,361 bp
2,322 bp
2,027 bp
564 bp
125 bp
48,502 bpTotal Size