Prelab Activity 1 a Review of Restriction Enzymes

Introduction

Special enzymes termed restriction enzymes have been discovered in many dissimilar bacteria and other unmarried-celled organisms. These restriction enzymes are able to scan along a length of Deoxyribonucleic acid looking for a particular sequence of bases that they recognize. This recognition site or sequence is mostly from four to 6 base pairs in length. Once it is located, the enzyme will attach to the Dna molecule and cut each strand of the double helix. The restriction enzyme will keep to do this forth the full length of the Dna molecule which will then break into fragments. The size of these fragments is measured in base of operations pairs or kilobase (1000 bases) pairs.

Since the recognition site or sequence of base of operations pairs is known for each brake enzyme, we can apply this to form a detailed analysis of the sequence of bases in specific regions of the Dna in which nosotros are interested.

In the presence of specific DNA repair enzymes, Deoxyribonucleic acid fragments will reanneal or stick themselves to other fragments with cut ends that are free to their ain end sequence. It doesn't thing if the fragment that matches the cutting end comes from the same organism or from a dissimilar i. This power of Dna to repair itself has been utilized by scientists to introduce foreign DNA into an organism. This DNA may contain genes that permit the organism to exhibit a new office or process. This would include transferring genes that will upshot in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area.

In this experiment, we will utilize restriction enzymes to cut up DNA from a modest virus chosen Bacteriophage λ. This virus is 48,502 base pairs in length which is very pocket-size compared with the homo genome of approximately 3 billion base pairs. Since the whole sequence of λ is already known we can predict where each brake enzyme will cutting and thus the expected size of the fragments that will be produced. If the virus Deoxyribonucleic acid is exposed to the restriction enzyme for only a brusk fourth dimension, then non every restriction site volition exist cut by the enzyme. This will result in fragments ranging in size from the smallest possible (all sites are cut) to in-between lengths (some of the sites are cutting) to the longest (no sites are cut). This is termed a partial restriction digestion.

In this experiment, we will perform a total restriction digestion. After overnight digestion, the reaction is stopped by add-on of a loading buffer. The Deoxyribonucleic acid fragments are separated by electrophoresis, a procedure that involves awarding of an electric field to crusade the Deoxyribonucleic acid fragments to migrate into an agarose gel. The gel is so stained with a methylene blue stain to visualize the DNA bands and may be photographed.

This laboratory will take approximately iii days. The restriction digestion takes place overnight and can be kept in the freezer until the adjacent course catamenia when it will be exist used for gel electrophoresis. The gels may be stained overnight prior to photographing or recording results.

Objectives

1. Empathize what a DNA brake enzyme is and how it works.
2. Learn to use a micropipette.
3. Learn to separate DNA on an agarose gel using electrophoresis.
4. Sympathize how to use a restriction digestion map to identify a sample DNA.
5. Compare the λ DNA bands on a gel to the known λ Dna restriction map.

Materials

For each lab group

• Four microtubes
• Microtube rack
• xx-µl micropipette (or 10-µl micropipette) and sterile tips
• Waterproof pen
• Chalice or foam cup with crushed ice for the following
  • twenty µl of 0.4 µg/µl λ DNA
  • ii.5 µl BamHI restriction enzyme
  • 2.5 µl EcoRI restriction enzyme
  • 2.five µl HindIII restriction enzyme
• 10 µl distilled h2o
• Gloves
• 500-ml chalice (solar day 2)
• Electrophoresis sleeping room (day ii)
• Power supply (day 2)
• xx µl 10X loading dye (day 2)
• 1.0% agarose gel (day 2)

Mutual Materials

• Container with TBE solution (1X)
• 37°C water bath west/ floating rack
• 60°C water bath or saucepan on a hot plate (day ii)
• Cooler with crushed ice
• Freezer (not frost-complimentary, if possible)
• Camera if desired
• Distilled h2o
• 0.002% methylene blueish stain (day 3)

Advance Preparation

Twenty-four hours 1:
1. If y'all saved the 1X TBE solution from the Gel Electrophoresis with Dyes activity, reuse it for this laboratory.
2. Obtain plenty crushed ice and ice containers (styrofoam cups) for each lab group.
3. Fill a pan with h2o and adjust it to 55°C on a hot plate
4. Fill a second pan with water and adapt it to 37°C on a hot plate while the students consummate preparation of the restriction digests.
5. Reconstitute the lambda Dna with sterile distilled water to 0.4 µg/µl.
6. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed.
7. Make the 1.0% agarose gel solution as follows:

   To make 100 ml of gel, which is sufficient for 3 gels, weigh out 1.0 thou of agarose and place into a 200- to 250-ml glass beaker or flask. Add 100 ml of 1X TBE (Tris-Borate-EDTA) buffer. Rut in the microwave for xxx seconds at a time, shaking gently each fourth dimension, until the agarose is completely melted. Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. Keep warm if the class will use information technology within a half 60 minutes. Otherwise, allow the solution to cool and solidify. Embrace and keep in the refrigerator.

   Twenty-four hours two:

8. Fill a pan with water and adapt information technology to 60°C.
9. Pour plenty agarose gels for each lab group as follows:
   • Wear gloves
   • Microwave or warm the agarose canteen in a hot waterbath until the gel liquefies. Exist sure to use a microwave designated for science purposes (non nutrient).
   • Firmly seal the ends of the gel tray using labeling tape.
   • Identify the plastic rummage in the slots close to the end of the tray.
   • Pour approximately 35-40 ml of agarose into each gel tray. This will effect in a thick gel and then that at least 20 µl of sample can be loaded into each well.
   • Permit cool until solidified (approximately 15 minutes).
   • If storing overnight, identify trays in a container or ziploc baggie with 0.5X TBE solution so they exercise not dry out.
   
   Day 3:
   
   
10. Remove student gels from the refrigerator.
11. Set containers for staining in a mutual area near a sink.

Note

Gels may be discarded in regular trash receptacle. A description of how to use a micropipet can be institute in Activity 2 - Gel Electrophoresis of Dyes.

Apply of Methylene Bluish:

Although methylene blue dye is not every bit sensitive every bit ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. Methylene bluish is non-toxic but will stain clothes, hands, and equipment, then always wear gloves. Use the stain close to a sink and clean upwards spills immediately. Utilise distilled or deionized water to de-stain gels. Merely use deionized water for making the 0.1X TBE buffer to make this stain since the high chlorine levels of most tap water will damage the Deoxyribonucleic acid. A single container of methylene blue dye should exist all that is needed since information technology may be reused several times and tending of down the sink.

Use of Power Supplies

See clarification in Gel Electrophoresis of Dyes - Activity 2
Enzymes

Restriction enzymes require special care for treatment and utilize. They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity.

Using proficient sterile technique, aliquot samples for students, existence careful to keep everything on ice until ready to be used.

Enzymes should be stored in a foam container in the freezer (not frost-free if available), along with the special buffer for each enzyme. The special buffers comprise the salt and pH requirements for optimal activity of each enzyme.

Lambda (λ) Dna:

The λ DNA used in this laboratory can exist equally either a linear or circular molecule, creating some confusion when interpreting brake digest results. By heating the sample to sixty°C for three minutes, immediately prior to electrophoresis, the hydrogen bonds holding the ends of the linear Deoxyribonucleic acid together in a circle volition be cleaved.

Background Reading

Since viruses have a relatively elementary genome, scientists have studied their Dna and used this information to examination theories and develop concepts that apply to the genetics of living organisms. Ane of the most studied viruses is called bacteriophage lambda (λ). Bacteriophage λ is a virus that infects bacterial cells.

Student Activity: Restriction Enzyme Assay - Methylene Blue stain

Background Reading

Bacteriophage λ is a virus that attacks bacterial cells and is one of the most studied viruses. The information from the relatively unproblematic virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms. The DNA of Bacteriophage λ is approximately 48,514 base pairs or 48.514 kilobase pairs in length while the human genome is approximately 3 billion base pairs.

This experiment uses special "restriction" enzymes that act as chemical scissors to cut λ Deoxyribonucleic acid into pieces. Each enzyme recognizes a unique sequence of 4-6 bases along the Dna strand and cuts the strand at these sites - the first step in a process chosen restriction mapping. These smaller, specific sections of an organism's Dna tin then be studied in detail and an outline of the whole genome tin be constructed. This procedure is i of the most of import in modern biology.

The pocket-sized fragments of Deoxyribonucleic acid are separated by gel electrophoresis. The move of the fragments will always exist towards the positive electrode because Dna is a negatively charged molecule. The fragments move through the gel at a charge per unit that is determined by their size and shape, with the smallest moving the fastest.

Deoxyribonucleic acid cannot be seen as it moves through the gel. A loading dye must be added to each of the samples earlier it is pipetted into the wells. The progress of the dye tin exist seen in the gel. It will initially appear as a bluish band, eventually resolving into ii bands of unlike colors.

The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a 300 base pair fragment of Deoxyribonucleic acid. The slower moving aqua band is xylene cyanol, about equivalent to a 9000 base pair fragment. The faster moving band must movement at to the lowest degree 4-7 cm from the wells to reach the all-time separation of DNA for analysis. Care should exist taken non to let the bromophenol blue band run off the end of the gel.

Following staining to locate the DNA, the gel is observed and the fragments appear as a pattern of bands. In this experiment, we will compare our banding pattern with a predicted issue shown in figure 1.

Figure 1. Lambda Dna Brake digest (Photo from J. Leach Laboratory)

Data may be provided past your instructor that details the process of isolating and analyzing these bands to create a DNA fingerprint.

Objectives

1. Understand what a Deoxyribonucleic acid restriction enzyme is and how it works.
2. Learn to use a micropipette.
3. Acquire to split up Deoxyribonucleic acid on an agarose gel using electrophoresis.
4. Sympathize how to use a restriction digestion map to identify a sample Dna.
5. Compare the λ DNA bands on a gel to the known λ Deoxyribonucleic acid restriction map.

Materials

For each lab grouping

• Four microtubes
• Microtube rack
• 20-µl micropipette and sterile tips
• Waterproof pen
• 250 µl distilled water
• Gloves
• xx µl 10X loading dye (mean solar day 2)
• 1.0% agarose gel (24-hour interval 2)
• Beaker or cream cups with ice for each of the post-obit:
  • 20 µl of 0.iv µg/µl λ Deoxyribonucleic acid - proceed in cup of ice
  • 2.five µl BamHullo restriction enzyme - continue in loving cup of ice
  • 2.5 µl EcoRI restriction enzyme - go along in loving cup of water ice
  • ii.v µl HindIII brake enzyme - keep in loving cup of water ice
• 500 ml beaker (day 2)
• Colored lab tape (mean solar day 2)

Common Materials

• Electrophoresis bedroom (twenty-four hour period 2)
• Power supply (day ii)
• Container with TBE buffer (1X)
• 37°C water bath w/floating rack
• 60°C water bath w/floating rack
• Cooler with crushed water ice
• Freezer (non-frost-free, if possible)
• Distilled h2o
• 0.002% methylene blue stain (day 3)
• Stain container (day iii)

Precautions

The methylene blue dye will stain skin, clothes, and equipment. Always habiliment gloves and safety glasses. Practice all staining in a central area near the sink.

Procedure

1. Put on gloves. Proceed all enzyme and Dna aliquots on ice through stride 6.
2. Label 4 microtubes, reagents as indicated below, and place them in the tube rack:

   Reagents	BamHI	EcoRI	HindIII	Control
   10X buffer	4 µl	4 µl	4 µl	4 µl
   Deoxyribonucleic acid	iv.0 µl	4.0 µl	iv.0 µl	4.0 µl
   BamHI	2.0 µl	0	0	0
   EcoRI	0	two.0 µl	0	0
   HindThree	0	0	2.0 µl	0
   Water	30.0 µl	30.0 µl	thirty.0 µl	32.0 µl

3. Prepare the micropipette to 4 µl and carefully add together iv µl of 10X restriction buffer to each tube. When adding the droplets of buffer to the restriction tube, touch the pipette tip to the bottom of the tube. Utilise a new tip for each buffer.
4. Gear up the micropipette to iv.0 µl and carefully add together 4.0 µl of DNA to each tube, using a new tip each time.
5. Add together 32.0 µl of distilled h2o to the command tube and 30.0 µl to the other reaction tubes.
6. Shut the microtubes and estrus in a 55°C waterbath for 10 minutes so immediately identify on ice for ii minutes.
7. Add ii µl of the advisable restriction enzyme to the reaction tubes as indicated on the grid. Use a new tip for each enzyme added.
8. Close the microtube caps and make sure that all the liquid is at the bottom of the tube past tapping the bottom of the tube gently on the desk top. Give the tubes to the teacher. They will exist incubated at 37°C overnight. The tubes volition and so exist frozen until the next grade (up to 2 months).

Solar day 2:
1. Put on gloves. Fill a styrofoam loving cup with ice, collect your DNA digestion tubes and keep on ice until needed.
2. The ane.0% agarose gel volition be placed into the gel box with the wells at the negative (black) end of the box.
3. Add approximately 150 ml of 1X TBE solution to the box so that the gel volition be covered with virtually 2 mm of buffer. Remove the comb past pulling straight up, making certain that the buffer covers the gel so that it will fill the wells and help them to retain their shape every bit the rummage is removed.
4. Estrus the microtubes in a 60°C water bathroom for iii minutes. This volition suspension whatever hydrogen bonds property the ends of the linear DNA together in a circumvolve.
5. Add 4 µl of loading dye to the bottom of each of the microtubes and eject the tip into the tube. Addition of the loading dye will also stop the restriction reaction taking identify in each tube. (The reaction can be stored in the refrigerator at this betoken for use at a subsequently date if necessary, in this case remove the tips and close the tube caps.)
6. Set up up the electrophoresis apparatus as described in Gel Electrophoresis of Dyes - Action two.
7. Load 20 µl of each sample into a well as shown in figure 2 in a higher place. Use the tips that were left in each tube or brand sure that you use a new tip for each sample if you stored the tubes overnight. Turn on the current for virtually 30-45 minutes. When the imperial dye from the loading dye is nearly 1 cm from the end of the gel, the ability supply should exist turned off and the gel box unplugged.
8. Place gel in a 0.002% methylene blue solution in 0.1X TBE and stain overnight at four°C or for 2 hours at room temperature.

Day 3:
1. Observe the gel over a white low-cal. If the bands are non visible considering of a high background staining, place the gel in 0.1X TBE with gentle agitation, irresolute the buffer every 30-60 minutes until y'all are satisfied with the degree of destaining. (from http://wheat.prisoner of war.usda.gov/~lazo/methods/lazo/met1.html)
2. Photograph if desired.
3. Wash the work area thoroughly to be sure that no stain solution is left in contact with surfaces. Wash your easily!
4. Complete the activeness sheet and appropriate forensics activities from either website beneath.

Pupil Activity

Restriction enzymes cut at specific sites along the Dna. These sites are determined past the sequence of bases which normally form palindromes. Palindromes are groups of letters that read the same in both the forward and backwards orientation. In the case of DNA the messages are found on both the frontwards and the reverse strands of the DNA. For instance, the 5' to three' strand may take the sequence GAATTC. The complimentary bases on the opposite strand will be CTTAAG, which is the same every bit reading the first strand backwards! Many enzymes recognize these types of sequences and will attach to the DNA at this site then cut the strand between two of the bases. The brake enzymes which nosotros used in this laboratory are EcoRI, HindIii and BamHi and their sequences are as follows, with the cut site indicated by the arrow.

This figure shows the size of each of the fragments/bands produced when λ Dna is cut with each of these restriction enzymes. The sizes were adamant past comparison to a molecular ladder which has bands of known sizes when it is separated by electrophoresis at the same time equally the digested λ DNA.

Brake sites of Lambda (λ) DNA - in base pairs (bp)

The sites at which each of the 3 different enzymes will cut lambda DNA are shown in the maps Enzymes A, B and C beneath.

1. Calculate the size the resulting fragments will be later on digestion and write them on the maps.
2. How many fragments would you expect to encounter for each of the maps A, B and C?
3. Depict these fragments onto the graph beneath.
4. Now compare the size of the fragments that you lot take calculated with the bands shown in the photographs of the gels and determine which of the enzymes, BamHow-do-you-do, EcoRI and Hind3 were used to cut A, B and C.
5. How many times does the sequence GAATTC occur in the λ DNA sequence? What about AAGCTT and GGATCC?
6. Are at that place as many bands in your gel equally you would expect to run across based on the results of your calculations? If the number is different explain what you remember has happened.

Answers to Educatee Activity

1. See map above
2. Under ideal conditions at that place would be 6 fragments from Enzymes A and B, and eight fragments from Enzyme C.
3. See students graph
4. Enzyme A = BamHI
   Enzyme B = EcoRI
   Enzyme C = HindIii
   Annotation: In not-platonic conditions, the enzyme may non cut at all sites, and a fractional restriction digestion will result
5. GGATCC is the recognition site for BamHI and is found in λ DNA at 5 locations.
   GAATTC is the recognition site for EcoRI and is establish in λ DNA at 5 locations.
   AAGCTT is the recognition site for HindIII and is constitute in λ DNA at 7 locations.
6. Sometimes bands that are very close together in size will not be visible separately on these gels. There may be a unmarried thicker band that indicates that two bands are co-localizing. When bands are very small-scale (500 bp or less) they may take run off the end of the gel and therefore no longer be nowadays.

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Source: https://www.apsnet.org/edcenter/disimpactmngmnt/labexercises/PlantBiotechnology/Pages/Activity3.aspx

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