Gel Electrophoresis lab write-up
Title: Restriction Digestion and Analysis of Lamba DNA
Objective:
1. To learn how to cut lamba DNA into a series of fragments using restriction enzymes.
2. To learn to separate and sort a large group of DNA molecules according to size
3. Know how to determine the size of each molecule separated by gel electrophoresis.
Materials:
• Ruler • 5µl pipette
• Marking pencil • 1µl pipette
• DNA from bacteriophage (lambda) • Disposable tips
• Three restriction enzymes (PstI, EcoRI, and • Mini centrifuge
HindIII.) • Agarose gel and holder
• Micro test tubes • Loading buffer containing the two dyes
• Restriction buffer (used in gel electrophoresis)
• Ice • Parafilm
• Water (room temp and ice temp) • Plastic wash bins
• Styrofoam holder
Lesson One: Introduction to Restriction Analysis
1. Describe any pattern you might see in the upper sequence of bases:
Answer: There is no specific type of pattern associated with the upper sequence of bases.
2. Compare the bases in the upper DNA strand to those in the lower strand. Can you discover any
relationship between the upper and lower strands? Describe it.
Answer: A always pairs with T; G always pairs with C.
3. Do you notice any grouping of bases that when read towards the right on the upper strand and read
toward the left on the bottom strand are exactly the same?
Answer: CTTAAG
4. How many base pairs are there to the left of the “cut”?
Answer: 4
5. How many base pairs are there to the right of the “cut”?
Answer: 10
6. Counting the number of base pairs, is the right fragment the same size as the left fragment?
Answer: No, it’s larger.
7. How could you describe the size of each fragment in terms of the number of base pairs in the fragment?
Answer: Fragment 1 is a 4-base-pair fragment.
Fragment 2 is a 10-base-pair fragment.
8. If the GAATTC palindrome is repeated 4 times on the same piece of linear DNA, and the restriction
enzyme that recognizes that base sequence is present and digests the DNA, how many DNA fragments
will be produced?
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, Answer: 5
9. If the GAATTC palindrome repeats are randomly found along the DNA strand, then what can you say
about the sizes of the fragments that will be produced when the DNA is digested with a restriction
enzyme that recognizes that sequence?
Answer: Random sized fragments will be produced.
Lesson One: Restriction Digestion (Lab Procedure)
1. Obtain micro test tubes that contain each enzyme solution, lambda DNA, and restriction buffer from the
common station. Keep all the stock solutions on ice.
2. Label four micro test tubes L, P, E, and H and place them in the foam Micro test tube holder.
L = Uncut lambda DNA (yellow tube)
P = PstI restriction digest of lambda DNA (violet tube)
E = EcoRI restriction digest of lambda DNA (green tube)
H = HindIII restriction digest of lambda DNA (orange tube)
• Describe the appearance of the DNA in solution.
Colorless
• Is the DNA visible?
No
4. You will set up your digests in micro test tubes. To each tube, add 4 μl of uncut lambda DNA, 5 μl of
restriction buffer and 1 μl of enzyme. Add only one kind of enzyme to a tube. Do not add enzyme
into the tube labeled “L”. Important note: First add DNA, then restriction buffer, and then the
enzymes to the tubes. Use a fresh pipette tip for restriction buffer and each enzyme.
Tube Lambda DNA Restriction Buffer PstI EcoRI HindIII
P 4 μl 5 μl 1 μl ---------- ----------
E 4 μl 5 μl ------------ 1 μl ----------
H 4 μl 5 μl ------------ ----------- ----------
L 4 μl 6 μl ------------ ----------- 1 μl
• In which tube do you expect no changes to occur—that is, no DNA fragments produced?
The DNA in the uncut lambda DNA tube, which gets no enzyme, should remain intact as a single band.
• What is missing in that tube that leads you to that decision?
No restriction enzymes are added to that tube, thus no digestion will occur, and no fragments will be
produced. This tube (L) is the control tube.
5. Tightly cap each tube. In order to mix all reagents, hold the top of a micro test tube between the index
finger and thumb of one hand and flick the bottom of the tube with the index finger of the other hand.
Gently tap the bottom of the tub on the table to collect the liquid. If you are using a centrifuge, place the
four tubes from your tube into the centrifuge, being sure that the tubes are in a balanced arrangement in
the rotor. Have your teacher check before spinning the tubes. Pulse-spin the tubes (hold the button for a
few seconds).
6. Let the sample tubes sit at room temperature over night.
This study source was downloaded by 100000831525611 from CourseHero.com on 02-02-2022 12:57:11 GMT -06:00
https://www.coursehero.com/file/8487102/Gel-Electrophoresis-Write-Up/
Title: Restriction Digestion and Analysis of Lamba DNA
Objective:
1. To learn how to cut lamba DNA into a series of fragments using restriction enzymes.
2. To learn to separate and sort a large group of DNA molecules according to size
3. Know how to determine the size of each molecule separated by gel electrophoresis.
Materials:
• Ruler • 5µl pipette
• Marking pencil • 1µl pipette
• DNA from bacteriophage (lambda) • Disposable tips
• Three restriction enzymes (PstI, EcoRI, and • Mini centrifuge
HindIII.) • Agarose gel and holder
• Micro test tubes • Loading buffer containing the two dyes
• Restriction buffer (used in gel electrophoresis)
• Ice • Parafilm
• Water (room temp and ice temp) • Plastic wash bins
• Styrofoam holder
Lesson One: Introduction to Restriction Analysis
1. Describe any pattern you might see in the upper sequence of bases:
Answer: There is no specific type of pattern associated with the upper sequence of bases.
2. Compare the bases in the upper DNA strand to those in the lower strand. Can you discover any
relationship between the upper and lower strands? Describe it.
Answer: A always pairs with T; G always pairs with C.
3. Do you notice any grouping of bases that when read towards the right on the upper strand and read
toward the left on the bottom strand are exactly the same?
Answer: CTTAAG
4. How many base pairs are there to the left of the “cut”?
Answer: 4
5. How many base pairs are there to the right of the “cut”?
Answer: 10
6. Counting the number of base pairs, is the right fragment the same size as the left fragment?
Answer: No, it’s larger.
7. How could you describe the size of each fragment in terms of the number of base pairs in the fragment?
Answer: Fragment 1 is a 4-base-pair fragment.
Fragment 2 is a 10-base-pair fragment.
8. If the GAATTC palindrome is repeated 4 times on the same piece of linear DNA, and the restriction
enzyme that recognizes that base sequence is present and digests the DNA, how many DNA fragments
will be produced?
This study source was downloaded by 100000831525611 from CourseHero.com on 02-02-2022 12:57:11 GMT -06:00
https://www.coursehero.com/file/8487102/Gel-Electrophoresis-Write-Up/
, Answer: 5
9. If the GAATTC palindrome repeats are randomly found along the DNA strand, then what can you say
about the sizes of the fragments that will be produced when the DNA is digested with a restriction
enzyme that recognizes that sequence?
Answer: Random sized fragments will be produced.
Lesson One: Restriction Digestion (Lab Procedure)
1. Obtain micro test tubes that contain each enzyme solution, lambda DNA, and restriction buffer from the
common station. Keep all the stock solutions on ice.
2. Label four micro test tubes L, P, E, and H and place them in the foam Micro test tube holder.
L = Uncut lambda DNA (yellow tube)
P = PstI restriction digest of lambda DNA (violet tube)
E = EcoRI restriction digest of lambda DNA (green tube)
H = HindIII restriction digest of lambda DNA (orange tube)
• Describe the appearance of the DNA in solution.
Colorless
• Is the DNA visible?
No
4. You will set up your digests in micro test tubes. To each tube, add 4 μl of uncut lambda DNA, 5 μl of
restriction buffer and 1 μl of enzyme. Add only one kind of enzyme to a tube. Do not add enzyme
into the tube labeled “L”. Important note: First add DNA, then restriction buffer, and then the
enzymes to the tubes. Use a fresh pipette tip for restriction buffer and each enzyme.
Tube Lambda DNA Restriction Buffer PstI EcoRI HindIII
P 4 μl 5 μl 1 μl ---------- ----------
E 4 μl 5 μl ------------ 1 μl ----------
H 4 μl 5 μl ------------ ----------- ----------
L 4 μl 6 μl ------------ ----------- 1 μl
• In which tube do you expect no changes to occur—that is, no DNA fragments produced?
The DNA in the uncut lambda DNA tube, which gets no enzyme, should remain intact as a single band.
• What is missing in that tube that leads you to that decision?
No restriction enzymes are added to that tube, thus no digestion will occur, and no fragments will be
produced. This tube (L) is the control tube.
5. Tightly cap each tube. In order to mix all reagents, hold the top of a micro test tube between the index
finger and thumb of one hand and flick the bottom of the tube with the index finger of the other hand.
Gently tap the bottom of the tub on the table to collect the liquid. If you are using a centrifuge, place the
four tubes from your tube into the centrifuge, being sure that the tubes are in a balanced arrangement in
the rotor. Have your teacher check before spinning the tubes. Pulse-spin the tubes (hold the button for a
few seconds).
6. Let the sample tubes sit at room temperature over night.
This study source was downloaded by 100000831525611 from CourseHero.com on 02-02-2022 12:57:11 GMT -06:00
https://www.coursehero.com/file/8487102/Gel-Electrophoresis-Write-Up/
