Plant Lab Protocols / Methodology for Nucleic Acids
Restriction of DNA
Recombinant DNA technology is a modern methodology in the field of molecular biology to manipulate genes. It includes enzymes to cut and form DNA molecules. The restriction endonucleases cut at the interior part of DNA. These enzymes are found in bacteria and in vivo are involved in recognition and destruction of foreign DNA. Invading phage DNA for instance will be restricted by such enzymes. The bacteria protect their own DNA by modification process.
The essential feature of restriction endonuclease action is that the enzyme recognizes a particular sequence of bases. Type II restriction enzymes cut the DNA within the recognized sequences. Each enzyme has its own characteristic recognition sequence and it may be 4 -7 bases long with dyad symmetry.
With the availability of nearly 50 restriction enzymes commercially, it is now possible to construct the physical map of genes after digestion of the DNA with different restriction enzymes and subsequent separation of DNA fragments on agarose gels.
Materials
» Plasmid DNA
» Plant DNA
» Lambda DNA
» Restriction Buffer (10x)
The following restriction buffer (10x) can be used with most restriction endonucleases.
100mM Tris-HCl pH 7.4, 100mM MgCl2,
100mM NaCl, 10 mM DTT (Refer Note 3)
» Stopping Mix
20% sucrose
10% Ficoll
10mM EDTA Na2
1% bromophenol blue
1% xylene cyanol FF
» Sterile Double Distilled Water
» Restriction Enzyme
» Microfuge Tubes (0.5mL)
» Pipetman (Gilson/Finnpipette 0-20mL)
Procedure
1.
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Label microfuge tubes 1-10 and arrange them open in a rack.
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2.
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Bring all the reactants on ice. DNA samples stored frozen are thawed quickly and brought on ice. Each reaction tube should contain about 1mg DNA. Restriction enzymes should be diluted before use to a concentration of one unit/mL. (One unit is defined as the enzyme activity which completely digests 1mg of DNA in one hour at 37°C.)
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3.
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Prepare the following reaction mixes by carefully pipetting into the bottom of microfuge tubes
Recipes (mL)
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Tube
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1
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2
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3
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4
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5
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6
|
7
|
8
|
9
|
10
|
11
|
12
|
l DNA
|
2
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2
|
2
|
2
|
|
|
|
|
|
|
|
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PlasmidDNA
|
|
|
|
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2
|
2
|
2
|
|
|
|
|
|
Plant DNA
|
|
|
|
|
|
|
|
2
|
2
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2
|
|
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Restriction buffer (10x)
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2
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2
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2
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2
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
2
|
Sterile water
|
16
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14
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14
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12
|
16
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14
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14
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16
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14
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14
|
|
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EcoRI
|
-
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2
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-
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2
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-
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2
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-
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-
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2
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-
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|
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Hind III
|
-
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-
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2
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2
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-
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-
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2
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-
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-
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2
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Total volume (mL)
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20
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20
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20
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20
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20
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20
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20
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20
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20
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20
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20
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20
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20mL in each tube
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|
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4.
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Mix contents of tube carefully. Centrifuge all the tubes in a microfuge (Eppendorf) for 2 sec to settle the contents at the bottom of tubes.
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5.
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Incubate at 37°C in a water bath for 60 min or longer if necessary.
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6.
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Terminate the digestion by adding 5mLof stopping mix to each tube or by heating at 65°C for 10 min.
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7.
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Mix and spin down as before.
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8.
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Load equal volume (15/20mL) of each digest in agarose minigel wells and run (see DNA electrophoresis in agarose gel).
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Notes
1.
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Siliconise the microfuge tubes so that no solution sticks to the side of the tubes. Prepare a 5% solution of dichlorodimethyl silane in chloroform, fill in the tubes and leave for a few minutes. Rinse the tubes with water thoroughly. Glassware can also be similarly siliconised and baked at 180°C overnight.
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2.
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Use a clean pipette tip (or microcapillary) for each transfer in order to avoid cross-contamination between solutions.
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3.
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Use the restriction buffer (10x) supplied along with the restriction enzymes for satisfactory results wherever possible. Each restriction enzyme requires specific ionic strength for optimum and precise cut. For most frequently used enzymes the restriction buffer (10x) composition is given below:
EcoRI and 100mM Tris-HCl pH 7.9, 60mM MgCl2
Sal I 1.5M NaCl, 60mM 2 MSH, 1mg/mL BSA (Fraction V)
Hind III 200mM Tris-HCl pH 7.5,70mM MgCl2,
500mM NaCl, 70mM 2-mercaptoethanol
Pst I 200mM Tris-HCl pH 7.5, 100mM MgCl2,
500mM (NH4)2SO4
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4.
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The restriction enzymes are usually supplied with glycerol as a stabilizing agent Note that if the concentration of glycerol exceeds 10% in the digestion mixture, the specificity of most enzymes is altered.
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5.
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The enzyme activity is considerably reduced even with proper storage after about six months.
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6.
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The reaction volumes are usually less than 50mL and contain about 1mg of DNA (More if a large number of fragments are expected to be generated).
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References
1. Maniatis, T, Fritsch, E F and Sambrook, J (1982) Molecular Cloning - A Laboratory Manual Cold Spring Harbour USA.
2. Manual on Techniques in Molecular Biology {Nucleic Acids) (1986) Workshop held at the Department of Biochemistry Tamil Nadu Agricultural University Coimbatore p 9.
3. Boffey, S A (1985) In: Experiments in Molecular Biology (Ed Slater, R J) Humana Press New Jersey p 7.
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