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The below mentioned article provides an Experimental Guide to Molecular Biology.
Molecular Biology # Experiment 1. Isolation of DNA from Cultured Cells:
Laboratory Methods:
The process involves a number of steps which are described separately:
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A. Preparation of Cell Pellet:
DNA can be made from cells growing in monolayer or in suspension culture, and, in general, 100 µg DNA will be obtained from 107 cells. Cells growing in monolayer can be harvested either with enzyme treatment (e.g. trypsin or collagenase) or by scraping the cells gently off the dish with a rubber policeman.
The latter treatment is easier and used for most purposes but the former treatment may be better for delicate primary cultures of cells. Cells growing in suspension in liquid or semisolid medium can be transferred to a centrifuge tube simply with a pipette. During harvesting, all cell preparations should be kept ice-cold.
Solutions and chemicals required:
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Phosphate-buffered saline (PBS)
Equipments recommended:
Pipettes
Rubber policeman
Sterile plastic centrifuge tubes (Falcon)
Bucket of ice Refrigerated centrifuge
Procedure:
1. Cells growing in monolayer culture are washed twice on the dish with PBS. They are then scraped off the dish in cold PBS with a rubber policeman, transferred to a sterile plastic centrifuge tube on ice and pelleted by centrifugation at 4°C at 1,000 g for 10 minutes.
2. Cells growing in suspension are transferred directly in cold PBS to a sterile plastic centrifuge tube on ice. The cells are pelleted by centrifugation at 4°C at 1,000 g for 10 minutes and the cell pellet washed twice with ice-cold PBS.
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3. The cell pellet can be used at once or stored frozen below -70°C.
B. Lysis of Cells:
The cells are lysed with detergent (SDS) and incubated with proteinase K to break down protein molecules.
Solutions and chemicals required:
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1 M Tris- HCl pH 7.4
0. 5.M EDTA pH 7.5
1 mg/ml proteinase K (Boehringer) in water.
This must be freshly made Distilled water
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10% (w/v) SDS (Serva) in water.
Equipments recommended:
Pipettes
Water bath at 37°C
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Procedure:
1. The cell pellet is re-suspended in an aliquot of the following solution, using 1.0 ml for 107cells:
1 M Tris-HCl pH 7.4 10 µl
0.5 M EDTA pH 7.5 20 µl
1 mg/ml proteinase K 100 µl
Distilled water 870 µl
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2. Add as fast as possible 50 µl of 10% SDS.
3. Incubated at 37°C in a water-bath for 4 hours.
C. Phenol Extraction:
The lysate is extracted with phenol. Since phenol and water are immiscible, the protein extracted into the phenol layer is removed from nucleic acids left in the aqueous layer.
Phenol used in this procedure must be very pure and should be redistilled before use, water-saturated and equilibrated with Tris buffer. Nowadays, redistilled phenol can be purchased from companies (such as Rathburn Chemicals) but any other phenol product must be redistilled before use.
Some years ago, isoamyl alcohol was added to the phenol to prevent frothing but this is no longer used for this protocol since use of chloroform appears to be adequate. The final washes with chloroform alone serve to remove all traces of phenol.
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Solutions and chemicals required:
5 M NaCl
Chloroform
Phenol (see below)
Preparation of phenol:
(i) Take 100 ml water-saturated redistilled phenol (Rathburn Chemicals).
(ii) Add 100 ml 0.5 M Tris-HCl pH 8.0 containing 1 mM EDTA.
(iii) Mix well and leave for 1 hour at room temperature.
(iv) Remove aqueous phase and discard.
(v) Repeat procedures (ii), (iii) and (iv) once more.
(vi) Add 100 ml 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA.
(vii) Mix well and leave 1 hour at room temperature.
(viii) Remove aqueous phase and discard.
(ix) Repeat procedures (vi), (vii) and (viii) twice more.
(x) Add 100 ml 10 mM Tris-HCl, pH 8.0
(xi) Store at 4°C in the dark.
With time, phenol will oxidize and change from colourless to orange. This can be slowed down by addition of 0.02% 8-hydroxyquinoline to the phenol. The solution can, however, be used for 6 months or more.
Phenol causes severe burns and must be handled with care.
Equipments recommended:
Agar pipettes (wide-bore)
Corex glass centrifuge tubes
Bench-top centrifuge at room temperature.
Procedure:
1. Add 30 µ1 5 M NaCl to each 1 ml of cell lysate.
2. Transfer to a Corex glass centrifuge tube.
3. Add an equal volume of phenol: chloroform (1: 1 v/v) mixture.
4. Seal tube and mix gently by inverting the tube.
5. Separate the phases by low-speed centrifugation (2,000 g for 10 min) at room temperature.
6. Remove the upper aqueous layer to a clean Corex glass centrifuge tube.
7. Repeat procedures 3-6 once more.
8. Add an equal volume of chloroform alone.
9. Seal tube and mix gently by inverting the tube.
10. Separate the phases by low-speed centrifugation (2,000 g for 10 min) at room temperature.
11. Remove the upper aqueous layer to a clean Corex glass centrifuge tube.
12. Repeat procedures 8-11 once more.
D. Ethanol Precipitation:
High-molecular-weight nucleic acid can be precipitated from an aqueous salt solution by the addition of ethanol. Nucleic acid free of protein will be a white fibrous material. Precipitation as a colourless gelatinous material indicates the presence of protein bound to the DNA.
Solutions and chemicals required:
100% ethanol
70% v/v ethanol in water
1 M Tris-HCl, pH 8.0
0. 5.M EDTA, pH 8.0 Distilled water.
Equipments recommended:
Clear plastic disposable tubes Shaking water bath at 37°C
Procedure:
1. To the final aqueous phase, add 2 volumes of 100% ethanol.
2. Swirl gently to mix and the nucleic acid precipitate should be visible at once.
3. Remove the precipitate by fishing it out on a pipette and wash by immersion in 70% ethanol and then 100% ethanol — for a few seconds each. It is preferable not to subject the precipitate to centrifugation as this will compact it and make re-dissolving in water later very difficult.
4. Allow the precipitate to dry in the air for a few minutes, until it is no longer soaked in ethanol but not until it is bone dry.
5. Re-dissolve in the following solution, using 1.0 ml for each 107 cells at the beginning:
1 M Tris —HCl pH8.0 10 µl
0.5 M EDTA pH 8.0 20 µl
Water 970 µl
If the DNA does not dissolve immediately it should be incubated at 37°C with gentle rocking overnight. Sometimes it can take as long as 2 days to dissolve.
E. Ribonuclease Treatment:
Treatment so far will have resulted in isolation of all high-molecular-weight nucleic acids. RNA is removed by treatment with ribonuclease.
Solutions and chemicals required:
Stock ribonuclease A 51 mg/ml in water
(stored at -20°C)
Stock ribonuclease T1 1,000 units/ml in water
(stock at -20°C)
10% (w/v) SDS (Serva) in water
Distilled water
Equipments recommended:
Water-bath or oil bath at 95°C
Water-bath at 37°C
Procedure:
1. Take 2 of stock ribonuclease A and 5 µl of stock ribonuclease T1 and add 200 µl water. Heat to 95°C for 5 minutes. This destroys any DNase contaminant.
2. Add 40 of the treated ribonuclease to each 1 ml of DNA.
3. Incubate at 37°C in a water bath for 2 hours.
4. Add 40 µl 5 M NaCl and 10 µl 10% SDS.
F. Final Steps:
The final steps involve a second round of proteinase K treatment, phenol extraction and ethanol precipitation.
Solutions, chemicals and equipments required:
Procedure:
1. To mixture at the end of protocol E, add 100 µ1 proteinase K solution (1 mg/ml).
2. Incubate at 37°C in a water-bath for 2 hours.
3. Repeat protocol C, steps 2-12, once.
4. Repeat protocol D, steps 1-4, once.
5. Re-dissolve the final DNA in the following solution, using 100 µl for each 107 cells at the beginning:
1 MTris —HCl pH8.0 1 µl
0.5 M EDTA pH 8.0 2 µl
Water 997 µl
The DNA solution should be viscous with a concentration of about 1 µg/µl.
If the DNA does not dissolve at once, it should be incubated at 37°C with gentle rocking for up to 2 days.
Molecular Biology # Experiment 2.
DNA Analysis by Restriction Enzyme Digestion and Southern Blotting:
DNA analysis using restriction enzymes and Southern blotting enables the presence and structure of genes in cells to be studied in some detail fairly simply and quickly. The principles involved are illustrated in Fig. 1.2. High-molecular-weight DNA is cleaved with one or more restriction endonucleases, which cut DNA in a very site-specific manner.
The resulting DNA fragments are then separated by size on agarose gel electrophoresis. The double-stranded DNA fragments are denatured in the gel and the single- stranded pieces can then be transferred to a sheet of nitrocellulose.
It is this transfer of electrophoretically- resolved DNA fragments to nitrocellulose filters which is known as ‘Southern blotting’ (Southern, 1975). This entails laying a sheet of nitrocellulose (which acts as a filter) on top of the gel and establishing a flow of buffer through the gel and the nitrocellulose filter.
The buffer carries the DNA fragments upwards from the gel to the nitrocellulose, where they subsequently bind. A single-stranded nucleic acid probe — specific for the gene under study — is then radiolabeled and hybridized to the filter.
This probe can be a purified RNA, DNA or cloned fragment of genomic DNA. Whichever is chosen, the labelled probe will hybridize to any DNA fragment on the filter which contains complementary nucleotide sequences.
Autoradiography of the nitrocellulose filter will reveal the position of each piece of DNA that contains any part of the probe used. By comparing the positions with radiolabeled polynucleotide markers of known length, the molecular size of each band can be estimated.
Several Parameters can be analysed from these Restriction-enzyme Patterns:
1. The number of copies of a gene can be estimated by comparing relative intensities of bands on the X-ray film.
2. By using different combinations of restriction enzymes and different parts of the probe, a precise ‘restriction-enzyme map’ can be established.
3. Variant genes sometimes differ from their normal counterparts by the loss or gain of certain restriction-enzyme sites or in the size of fragments generated.
Such alterations can occur within either the structural gene or surrounding control areas, and can either be related directly to the abnormality or result from a polymorphism with linkage to the abnormal gene. Such variations have already proved useful in the diagnosis of certain clinical syndromes.
The major steps as involved are described and represented:
A. Digestion of DNA with Restriction Endonucleases:
The first step involves cleavage of high molecular weight DNA with one or more restriction endonucleases. These enzymes are now available from many commercial sources.
Equipments recommended:
Micro centrifuge tubes (1.5 ml volume)
(autoclaved) (Treff Lab)
Water-bath
Solutions and chemicals required:
DNA prepared in water.
10 × restriction enzyme (10 × RE) buffer,
Made as follows:
2 ml 1 M Tris-HCl pH 7.4 (autoclaved )
1.6 ml distilled water (autoclaved)
0.4 ml 1 M magnesium chloride (autoclaved)
28 µl stock β-mercaptoethanol
(This can be stored at -20°C for up to one month)
Restriction-enzyme preparation (Boehringer or Bio labs)
5 M sodium chloride (autoclaved)
Distilled water (autoclaved)
Procedure:
Set up the restriction-enzyme reaction mixture in a 1.5 ml micro centrifuge tube:
1. Add up to 20 µg of DNA, DNA solutions are viscous and should be measured with a pipette tip cut to give wider bore (about 2 mm).
2. Add 5 ml of 10 × RE buffer.
3. Add the required amount of enzyme (up to 5 µl volume only). The amount of enzyme required is calculated in units (1 unit of enzyme cuts 1 µg of DNA in 1 hour at 37°C in a volume of 50 µl).
4. Add 5 M NaCl as needed by the enzyme.
There are three groups of enzyme:
Low-salt enzymes — add 0.05 ml 5 M NaCl
Medium-salt enzymes — add 0.5 ml 5 M NaCl
High-salt enzymes — add 1.0 ml of 5 M NaCl
The salt requirements are specified by the supplier.
5. Add distilled water to make the volume up to 50 µl. The ideal volume for the reaction is 50 µl. However, larger volumes can be used if more than 20 µg DNA is to be cut or if the enzyme is so diluted that more than 5 µl has to be used. It should be remembered that the volume of enzyme used should never exceed one-tenth of the reaction volume.
6. Incubate reaction mixture for 1 hour minimum. Most enzymes work best at 37°C but some require other temperatures. This will be specified by the supplier. Theoretically, 1 hour should be adequate, but it is often both convenient and advantageous to leave the reaction overnight.
B. Ethanol Precipitation:
After restriction-endonuclease digestion, the samples are precipitated with ethanol. This serves to remove salts which might interfere during subsequent gel electrophoresis and also to concentrate the DNA into a smaller volume for loading onto the gel.
Solutions and chemicals required:
5 M sodium chloride (autoclaved)
100%, 95%, 70% ethanol (v/v in water)
Distilled water (autoclaved)
Equipments recommended:
Flask of dry ice
Micro centrifuge (Eppendorf or M.S.E.)
Spectrophotometer
Procedure:
1. Make 0.1 M with NaCl [i.e. add 1 µl of stock 5 M NaCl (autoclaved) to each 50 µl of digest].
2. Add 2.5 vol. of 95% ethanol.
3. Leave in dry ice for 30 minutes.
4. Spin in micro centrifuge at 4°C for 10 minutes.
5. Pour off ethanol and drain well from DNA pellet.
6. Add 0.5 ml of 70% ethanol (dissolves and removes NaCl).
7. Spin in micro centrifuge at 4°C for 10 minutes.
8. Again pour off ethanol and then add 0.5 ml of 100% ethanol (to dry).
9. Spin in micro centrifuge at 4°C for 10 minutes.
10. Pour off ethanol. Dry DNA pellet in air.
11. Take up DNA in distilled water (so that DNA is about 1 µg/µl).
12. Measure concentration of DNA:
Take 2 µl of the DNA and add to 500 µl of water. Read optical density at 260 nm (OD260).
A solution of DNA in water 1 mg/ml gives an OD260 of 20.
Thus, µg/µl.1 in DNA solution = 1/20 × OD260 × 250.
C. Prepare and run an Agarose Gel for DNA:
The DNA fragments produced from restriction-enzyme digestion are separated by size on an agarose gel.
Solutions and chemicals required:
5 × TBE buffer, made as:
106 g Tris base
55 g Boric acid
9.3 g di-potassium EDTA.
Dissolve and make up to 2 litres with water 10 mg/ml ethidium bromide (BDH) in water (store in the dark)
Agarose powder (SEAKEM-ME) (FMC Corp.) DNA sample buffer (DNA SB) made as:
330 µl glycerol
75 µl.1 5 × TBE buffer
250 µl 0.5 M EDTA (pH 7.0)
Mix thoroughly, then
Add 27 µl of 10% w/v SDS (Serva) in water (if this is added earlier it precipitates out)
Add bromophenol-blue powder (spatula tip, approx. 100 µg)
Equipment recommended:
Microwave oven or boiling water-bath
Agarose gel electrophoresis equipment (commercially available or home-made)
Electrophoresis power supply
Procedure:
1. Prepare mould for the gel. Many systems are available commercially for pouring gels but a simple home-made system is illustrated in Fig. 1.3. A Perspex plate is levelled using a spirit level.
A glass plate (14 cm × 19 cm) is used as the mould. Its edges are sealed with PVC electrical tape (or radioactive/biohazard-labelled tape) so that it forms a wall 1 cm high all around the plate. This is placed on the level Perspex plate. A simple Perspex comb is assembled at one end of the plate so that there is about 1 mm of space between the base of the teeth and the glass plate.
2. Weigh 0.8 g of agarose into a 200 ml conical flask. Add 100 ml of 1 × TBE buffer. Add 100 µl ethidium bromide solution (10 mg/ml in water). Heat in microwave oven or boiling water-bath until agarose dissolves.
3. Pour agarose onto gel mould and allow to set (1-2 hours).
N.B.:
The recipe given here provides a 0.8% agarose gel, which is useful for most purposes. To separate very large or very small DNA fragments, the concentration can be varied (For further details see Maniatis et al., 1982.).
4. The gel tank is prepared and connected to an electrophoresis power supply. Many systems are available commercially but a simple home-made system is illustrated in Fig. 1.4.
5. Place 1,600 ml of 1 × TBE buffer into the gel tank and add 2 drops of ethidium bromide solution (10 mg/ml).
6. Put the gel into the tank. The surface of the gel should be just submerged (by about 1 cm depth of buffer).
7. Make DNA samples up to 20 µl in water and add 8 of DNA SB.
8. Load DNA into wells of the gel.
Polynucleotides of known size can be loaded as required in a separate well, to provide
9. A fast test gel should be run initially to assess if the DNA is well cut:
Load 1 µg of DNA per well. Run 120 V (50 mA) 2-4 hours.
If DN A is not cut to completion, it must be re-digested with more enzyme (return to protocol A). If DNA is cut, the fragments can be separated on a main gel for Southern blotting.
This would be run as:
Load up to 20 µg DNA per well.
Run 25-30 V (20 mA) 18 hours.
All samples are run from the negative to the positive electrode.
D. Southern Transfers (blotting):
This procedure was first described by Southern (1975). It involves transfer of the electrophoretically resolved DNA fragments from the agarose gel onto a nitrocellulose filter.
Solutions and chemicals required:
1 M sodium hydroxide 5 M sodium chloride
1 M Tris — HCl pH 7.4 Distilled water
10 × SSC solution made as:
87 g sodium chloride
44.1 g trisodium citrate dehydrate
Dissolve in 800 ml distilled water
Adjust pH to 7.0 with 5 M NaOH
Make volume up to 1 litre with water
Nitrocellulose. This is available from many commercial sources
(recommended: Sartorius filters type SM 26; pore size 0.1 pm).
Equipments recommended:
Ultraviolet light
2 plastic trays (25 cm × 30 cm × 5 cm high) 2 large rubber bungs (size 49)
2 glass plates (14 cm × 19 cm)
Whatman filter paper (No. 1 and No. 3 mm)
One packet of paper hand towels
1 kilogram weight (e.g. a 500 ml bottle filled with water)
Clingfilm
Vacuum oven
Procedure:
1. DNA in the gel is stained with the ethidium bromide in the buffer and can be visualized under ultraviolet light. The DNA bands in the agarose gel are thus examined firstly under ultraviolet light, and photographed for a permanent record if a camera is available.
2. Place the gel in a plastic tray of convenient dimensions.
3. Denature the DNA into separate strands by using 2 × 30 minute washes of 250 ml each of the solution made as indicated:
250 ml 1 M NaOH
100 ml 5 M NaCl
150 ml distilled water
4. Neutralize the gel with 2 × 30 minute washes of 250 ml each of the solution made as indicated:
250 ml 1 M Tris — HCl pH 7.4 250 ml 5 M NaCl
5. Soak the gel in 10 × SSC solution for a few minutes.
6. The blot is set up as shown in Fig. 1.5
(a) Wet the nitrocellulose filter in distilled water in a plastic tray, then transfer to 10 x SSC solution.
(b) In another plastic tray, place two large rubber bungs on the bottom.
(c) Place a glass plate (14 cm x 19 cm) on top.
(d) Now place a sheet of 3 mm Whatman paper over the glass plate so that it acts as wick on either side of the plate.
(e) Add 600 ml of 10 × SSC solution to the tray and soak the Whatman paper, making sure that all air bubbles are removed.
(f) Carefully place the gel on top of the Whatman paper, again removing all air bubbles.
(g) Now cover all edges of the gel using four pieces of Clingfilm, tucking excess under the tray but taking care not to cover any area of the gel containing DNA. This ensures that the 10 x SSC solution passes evenly through the gel.
(h) Lay the wetted nitrocellulose sheet onto the gel surface, taking care to exclude air bubbles.
(i) Lay three sheets of presoaked (10 × SSC) Whatman No. 1 paper over the filter.
(j) Place about 8 cm of dry Kleenex Hi-Dri paper towels over the Whatman No. 1 paper and place a glass plate on top of this. Now put a kilogram weight on top and leave to blot overnight (a minimum of 18 hours).
7. Disassemble blot the next morning.
Place the filter (TAKE CARE: with DNA upwards) on dry tissues.
Leave to dry in air for about 30 minutes.
Check the gel under ultraviolet light to ensure that all the DNA has transferred.
Place the dried filter in a small envelope of filter paper (Whatman No. 3 mm) and bake in a vacuum oven at 80°C for 3-5 hours.
E. To make 32P-labelled Probe by Nick Translation of DNA:
Radiolabeled probes for nucleic acid hybridization can be prepared by a variety of methods, which are discussed in more detail elsewhere (Arrand, 1985). In this protocol, one reliable method only is presented. This involves labelling the probe by a nick translation reaction (Rigby et al., 1977). The enzyme DNase 1 is used to create single-strand nicks in double-stranded DNA.
Then the 5′ – 3′ exonuclease and 5′ – 3′ polymerase actions of E. coli. DNA polymerase 1 are used to incorporate radiolabeled deoxyribonucleotides while repairing the nicks.
Finally, the DNA is denatured with sodium hydroxide and free unincorporated deoxyribonucleotides are separated from the DNA by column chromatography. In practice, the end result of hybridization is usually cleaner if DNA used in this reaction is a piece of DNA under 2,000 bases in length and separated from any plasmid vector used for cloning.
Solutions and chemicals required:
Distilled water (autoclaved)
10 × nick translation (10 × NT) buffer, made as:
500 µl 1 M Tris-HCl pH 8.0 (autoclaved)
100 µl 0.5 M magnesium chloride (autoclaved)
10 µl β-mercaptoethanol
390 µl distilled water (autoclaved)
20 µ M dATP, dGTP and dTTP, made as:
Each deoxyribonucleotide triphosphate is dissolved in sterile distilled water to give a concentration of 10 mM. The pH of each solution is then adjusted to pH 7.0 using a solution of 0.05 M Tris base.
The concentration of each solution is checked by reading the optical density of a small aliquot diluted in water (the molar extinction coefficient for the bases is:
A 1.54 × 104 M-1 cm–1 at 259 nm;
G 1.37 × 104 M-1 cm–1 at 253 nm;
T 7.4 × 103 M-1 cm–1 at 260 nm).
These 10 mM stock solutions can be stored at -20°C and diluted in water for use at 20 pM concentration.
32P-dCTP purchased from Amersham (PB 10205) DNA probe.
1 mg/ml bovine serum albumin (BSA) (Sigma, Fraction V powder) in autoclaved distilled water DNase 1 (Worthington). A stock solution of 100 µg/ml can be stored at -20°C. This should be diluted to 100 µg/ml for use but, at this lower concentration, continual thawing and refreezing is not recommended. Aliquots should be stored frozen and thawed once only before discarding.
DNA polymerase I (Boehringer)
5 M sodium hydroxide (autoclaved)
G50 column buffer, made as:
10 ml 5 M sodium chloride (autoclaved)
5 ml 1 M Tris-HCl pH 7.4 (autoclaved)
5 ml 10% w/v SDS (Serva) in water (filter sterilised)
480 ml distilled water (autoclaved)
Sephadex G50 fine (Pharmacia) pre-swollen in the G50 column buffer
Equipments recommended:
32P-dCTP emits beta-radiation and should be handled with care:
All work should be done behind Perspex screens (1 cm thick) to protect the operator from radiation. Usual precautions for handling 32P-labelled chemicals must be observed at all times.
Geiger counter (for beta-emission)
Micro centrifuge tubes 1.5 ml volume (autoclaved) (Treff Lab)
Water-bath at 14°C (use a water-bath in a cold room)
5 ml sterile disposable plastic pipette
Siliconised sterile glass wool
Sterile tubing to set up a chromatography column
Scintillation counting facilities
Procedure:
Prepare nick translation mix in a 1.5 ml micro-centrifuge tube (this should be done behind Perspex screens):
1. 5 µl dATP (20 µM).
2. 5 µl dTTP (20 µM).
3. 5 µl dGTP (20 µM).
4. 5 µl 32P-dCTP (50 µ Ci/reaction).
5. 0.5 pg DNA (volume 0.5 µl).
6. 24.5 µl water.
7. 5 µl 10 × NT buffer.
8. Add 1 µl of I mg/ml BSA to stabilise the DNase which is added later.
9. Add 2.5 µl of DNase I (100/ng/ml). Leave at room temperature for 3 minutes.
10. Add 1 µl of DNA polymerase I.
11. Leave at 14°C for 2 hours.
12. Add 3.3 µl of 5 M NaOH to the mix, and leave at room temperature for 10 minutes.
13. Separate 32P-labelled DNA from 32P- dCTP on a column of Sephadex G50 fine.
(a) Prepare a column of Sephadex G50 fine in a disposable 5 ml pipette plugged with siliconised sterile glass wool. All column equipment should be sterile. Equilibrate the column with column buffer.
(b) Apply the reaction mix to the column and elute with column buffer. Monitor effluent with a Geiger Counter.
(c) Two peaks are formed on the column. The first peak is the labelled DNA. The second peak is 32P-dCTP. The first peak is collected in a volume of 1-2 ml.
(d) Count a 5 µl aliquot by liquid scintillation counting to estimate the specific activity (cpm/µg) of the DNA.
F. Hybridization:
Single-stranded DNA immobilized on the nitrocellulose filter is hybridized to the single stranded radiolabeled DNA probe. Several methods are available for this reaction but, commonly, formamide or dextran sulphate are used. Dextran sulphate, although viscous to handle, provides a reliable and sensitive assay method and is the method of choice described here.
The procedure described here provides high stringency and only closely homologous DNA sequences will hybridize. If the DNA probe is unlikely to be closely related to the DNA under examination, the stringency can be lowered by either lowering the temperature or increasing the salt concentration (i.e. amount of 20 x SSC solution added).
Equipments recommended:
Plastic bags and a plastic-bag sealer (any commercially available system sold for domestic use is suitable)
An incubator set at 65°C with shaking facilities.
(If this is not available, bags can be submerged in a shaking water-bath.)
Solutions and chemicals required:
Distilled water
20 × SSC solution made as:
174 g sodium chloride
88.2 g trisodium citrate dihydrate
Dissolve in 800 ml distilled water
Adjust pH to 7.0 with 5 M NaOH
Make volume up to 1 litre with water
25 × Denhardt’s solution, made as:
2 g bovine serum albumin (BSA) (Sigma, Fraction V)
2 g Ficoll 400 (Pharmacia)
2 g polyvinyl pyrolidone (Sigma)
Make up to 400 ml with water
Filter-sterilize by passing through a 0.45 µm filter
10% w/v SDS (Serva) in water. Sodium dodecylsulphate (Serva) is dissolved in water (10 g/ 100 ml) and filter-sterilized (0.2 µm)
Single-stranded (SS) DNA solution, made as:
1 mg/ml calf thymus DNA (Sigma); dissolve in boiling water,
Sonicate (amplitude low, 1 minute), then boil again;
Store in a sterile bottle at 4°C
50% dextran sulphate solution, made as:
500 g (Pharmacia sodium salt), plus 800 ml water
Shake occasionally by hand over several days to dissolve
Make volume to 1 litre with water
Procedure:
1. Prepare solution A:
3 ml 20 × SSC solution
8 ml 25 × Denhardt’s solution
9 ml distilled water
= 20 ml
Place the nitrocellulose blot inside a plastic bag. Add solution A. Expel air bubbles. Seal bag. Incubate for 1 hour at 65 °C with vigorous shaking.
2. Prepare solution B:
Cut off the corner of the plastic bag. Pour out solution A. Add solution B. Reseal. Incubate for 1 hour at 65°C with vigorous shaking.
3. Prepare solution C:
Cut off the corner of the plastic bag. Pour out solution B. Add solution C. Reseal. Incubate for 1 hour at 65°C with vigorous shaking.
4. Prepare solution D:
This is identical to solution C but contains in addition 5-10 million cpm of 32P-labelled DNA probe. Cut off the corner of the plastic bag. Pour out solution C. Add solution D. Reseal. Incubate for 15-18 hours at 65°C with vigorous shaking.
G. Washing the Blot:
After hybridization the Southern blot is washed to remove nonspecific hybridization. The important step in this procedure is step 3, which determines the stringency required. The conditions described here provide a high stringency wash.
If the DNA probe is not closely related to the DNA under examination, the stringency of the wash in step 3 should be lowered by either decreasing the temperature or increasing the salt concentration (i.e. amount of 20 x SSC added).
Solutions and chemicals required:
20 × SSC solution made as:
174 g sodium chloride
88.2 g trisodium citrate dihydrate
Dissolve in 800 ml distilled water
Adjust pH to 7.0 with 5 M NaOH
Make volume up to 1 litre with water
25 × Denhardt’s solution, made as:
2 g bovine serum albumin (Sigma)
2 g Ficoll 400 (Pharmacia)
2 g polyvinyl pyrrolidone (Sigma)
Make up to 400 ml with water
Filter-sterilize at 0.45 (am
10% w/v SDS (Serva) in water and filter-sterilized at 0.2 µm
Distilled water
Equipments recommended:
An incubator set at 65°C with shaking facilities (if this is not available, washing can be done in a shaking water-bath)
Plastic tray
Clingfilm
Whatman filter paper (No. 3 mm)
X-ray film cassette
X-ray film (recommended: Kodak XAR5)
Facilities for developing X-ray film
Procedure:
1. Remove the nitrocellulose filter from the plastic bag in which hybridization has taken place and put the filter into a plastic tray.
2. Wash at 65°C with 2 × 10 minute followed by 2 × 30 minute washes, 100 ml each time of solution made as indicated:
20 ml 20 x SSC solution
160 ml 25 × Denhardt’s solution
4 ml 10% SDS solution
216 ml distilled water
= 400 ml
3. Wash at 65°C with 2 × 30 minute washes of 100 ml each of solution made as indicated:
2 ml 20 × SSC solution
2 ml 10% SDS solution
196 ml distilled water
= 200 ml
4. Wash the blot with 4-5 rinses at room temperature (1 minute each) with 100 ml each of solution made as indicated:
75 ml 20 × SSC solution
425 ml distilled water
= 500 ml
5. While the blot is still wet it should be scanned with a Geiger counter. If a large amount of radioactivity is detected scattered all over the filter, there may be nonspecifically hybridized DNA still bound and the blot may need to be washed for longer (repeat steps 3 and 4).
6. After washing, allow the blot to air dry. Then place on 3 mm Whatman paper and cover in Clingfilm to prevent the X-ray film sticking to the blot. Expose to X-ray film.
Molecular Biology: Experiment # 3:
Most DNA sequences used as probes for Southern and Northern blotting will have been isolated and cloned into a plasmid vector. No attempt is made here to discuss any of the strategies for gene cloning. However, it is necessary to prepare large quantities of the DNA for use in the nick translation reaction and then as a probe — and it is these methods which are described here.
Plasmids are extra-chromosomal genetic elements found in a variety of bacterial strains. They are double- stranded closed circular DNA molecules, ranging in size from 1 kb to 200 kb, and they contain genes coding for enzymes that — under certain conditions — are advantageous to the bacterial host.
The most common phenotype used in cloning is that of resistance to antibiotics. The cloned gene will have been inserted into the plasmid at a specific restriction-enzyme site (which must be known). General features of the plasmid structure are given in Fig. 1.6.
Plasmid DNA is amplified in the bacteria. Thus the first step is to put the plasmid DNA into bacterial cells, a process known as transformation. The bacteria are treated with calcium chloride, which makes some of the cells temporarily permeable to small DNA molecules (Mandel and Higa, 1970; Cohen, Chang and Hsu, 1973).
Use of the new phenotype (usually resistance to the antibiotics ampicillin or tetracycline) conferred on the recipients by the plasmid allows simple selection of the bacteria which have been successfully transformed.
The bacteria are then left to grow for some hours, during which time the plasmids also replicate within the cells, giving many copies per cell. The bacteria are then harvested and lysed, and plasmid DNA is isolated from the bacterial host cell DNA.
Initially, a differential precipitation step is performed in which the large strands of bacterial genomic DNA — entangled in the remnants of lysed cells — are preferentially removed from the lysate.
Final purification of the plasmid is on caesium chloride gradients. The DNA is centrifuged to equilibrium in the gradient in the presence of saturating quantities of the intercalating dye, ethidium bromide. Plasmid DNA, which is covalently closed circular DNA, binds much less of the dye than the linear bacterial genomic DNA and, hence, bands at a higher density in the caesium chloride gradient (Radloff et al., 1967).
The purified plasmid is then ready for use in a nick translation reaction. In practice, however, final autoradiographs of Southern or Northern blots have lower backgrounds if the radiolabeled probe contains only the cloned sequences of interest and no plasmid sequences.
Thus, finally, the plasmid is cut with the appropriate restriction enzyme used for the cloning originally. This releases the cloned gene from the plasmid and the two DNAs can be separated by agarose gel electrophoresis. The principles involved in these methods are illustrated in Fig. 1.7.
The major steps are:
A. Transformation of Bacteria with Plasmid:
The bacteria are treated with calcium chloride, which makes some of them temporarily permeable to plasmid molecules. The bacteria are then grown overnight in agar containing either ampicillin or tetracycline.
Using the gene for antibiotic resistance contained in the plasmid, simple selection is- achieved of the bacteria that have been transformed. It is necessary to know for each plasmid which antibiotic resistance is operative.
Solution and chemicals required:
Bacto-agar
L-broth made as:
10 g Bacto-tryptone
5 g Bacto-yeast extract
10 g sodium chloride
Make volume to 1 litre with distilled water
Adjust pH to 7.5, if necessary with sodium hydroxide
Autoclave
Appropriate antibiotic
i. ampicillin 50 mg/ml in water (filter-sterilized through 0.2 µm filter)
ii. tetracycline 15 mg/ml in methanol (filter-sterilized through 0.2 µm filter)
2 M calcium chloride (filter-sterilized through 0.2 µm filter)
Distilled water (autoclaved)
TEN buffer made as:
1 ml Tris-HCl pH 7.4
0.2 ml 0.5 M EDTA
4 ml 5 M sodium chloride
Make up to 100 ml with distilled water
Filter-sterilize through 0.2 µm filter
Bacterial cells strain HB101
1 µg of plasmid DNA
Equipments recommended:
Balance for weighing
Autoclave
Bunsen burner
Sterile pipettes
Sterile pipette tips
Boiling water-bath or microwave oven
9 cm diameter bacteriological petridishes
Sterile 50 ml glass conical flasks
Sterile 250 ml glass conical flasks
Plastic sterile 50 ml tubes
Plastic sterile 2 ml tubes (cryotubes)
Shaking incubator at 37°C
Static incubator at 37°C
Water bath at 37°C
Bucket of ice
Glass spreader (made by bending a Pasteur pipette in a Bunsen flame at an angle of 90°, to give a flat glass surface about 5 cm long)
Beaker with about 50 ml ethanol
Refrigerated Sorvall centrifuge at 4°C with rotor SS34 (or equivalent)
Procedure:
1. Prepare agar plates as:
(i) Weigh 2.2 g bacto-agar into a 500 ml bottle,
(ii) Add 200 ml of L-broth,
(iii) Autoclave the mixture,
(iv) Boil in a water-bath or heat in microwave oven until liquid,
(v) Cool 50°C and add antibiotic solution (0.2 ml of ampicillin or tetracycline stock solution),
(vi) Pour 30 ml onto each of six bacteriological petridishes (9 cm size),
(vii) Flame the surface of the agar in the petridishes with a cool Bunsen flame to remove any air bubble,
(viii) Leave at room temperature for 24 hours to set and dry out.
2. Prepare stock culture of bacteria as:
(i) Place 5 ml of L-broth in a sterile 50 ml glass conical flask,
(ii) Add about a hundred bacterial cells strain HB101.
(iii) Shake vigorously in an incubator at 37°C overnight.
3. Prepare bacteria for transformation as:
(i) Place 0.5 ml of the overnight bacterial culture in a 250 ml glass conical flask,
(ii) Add 50 ml of L-broth.
(iii) Shake vigorously in an incubator at 37°C for 2 hours until the OD600 is about 0.5.
(iv) Transfer to a plastic sterile 50 ml tube.
(v) Pellet the bacteria in a Sorvall centrifuge (rotor SS34) at 4°C at 10,000 rpm (12,000 g) for 5 minutes.
(vi) Re-suspend bacteria in 50 ml of 80 mM calcium chloride (made by using 2 ml 2 M calcium chloride and 28 ml water),
(vii) Leave on ice for 15 minutes,
(viii) Pellet bacteria by centrifugation at 4°C as at step (v).
(ix) Re-suspend bacteria in 5 ml of 80 mM calcium chloride (made by using 0.2 ml 2 M calcium chloride and 4.8 ml water).
(x) Keep on ice and use within 4 hours.
4. Transform bacteria with plasmid as:
(i) Place 200 µl of treated bacteria in a sterile 2 ml tube,
(ii) Add 1 µg of plasmid made up to 100 with TEN buffer,
(iii) Leave on ice for 30 minutes,
(iv) Place in a water-bath at 37°C for 5 minutes.
(v) Add 300^1 of L-broth.
(vi) Leave in a water-bath at 37°C for another 30 minutes (for tetracycline resistance) or another 1 hour (for ampicillin resistance).
5. Plate transformed bacteria onto agar plates containing antibiotic:
(i) Prepare agar plates with different numbers of bacteria: e.g. place 5 µl onto one dish, 50 onto another and 500 µl onto another.
(ii) Spread bacteria over each dish evenly using a sterile glass spreader. The spreader is sterilized by dipping it in a beaker of ethanol and lighting in a Bunsen flame,
(iii) Incubate the petridishes upside down in an incubator at 37°C overnight until bacterial colonies are visible by eye.
B. Growth of Bacteria and Amplification of Plasmid:
A single bacterial colony is picked off an agar dish and is amplified in suspension, firstly as a small 5 ml culture and then to much larger volumes.
Solutions and chemicals required:
L-broth (made as in protocol A) Appropriate antibiotic
i. ampicillin 50 mg/ml in water (filter-sterilized through 0.2 µm membrane)
ii. tetracycline 15 mg/ml in methanol (filter-sterilized through 0.2 µm filter)
Glycerol (autoclaved)
Equipments recommended:
Platinum wire loop on a handle
Bunsen burner
Sterile 50 ml glass conical flasks
Sterile 1-litre glass conical flasks
Shaking incubator at 37°C
Procedure:
1. Place 5 ml of L-broth in a sterile 50 ml glass conical flask.
2. Add 5 µl of stock ampicillin or tetracycline solution.
3. Pick a single bacterial colony off an agar dish with a sterile platinum loop and add to the L-broth. (The wire loop is sterilized by heating in a Bunsen flame until it is red hot; it must be cooled in air before touching the bacteria.)
4. Shake vigorously in an incubator at 37°C overnight.
5. Next morning, place 400 ml of L-broth in a sterile 1-litre glass conical flask.
6. Add 1.0 ml of stock ampicillin or tetracycline solution.
7. Add 4 ml of the bacterial culture.
8. Shake vigorously in an incubator at 37°C overnight.
9. Next morning, store a 2-ml aliquot of the bacterial cells mixed with 2 ml of glycerol at – 20°C as a stock solution for any future use. Such cells can be removed from the freezer and a small (0.1 ml) aliquot amplified whenever needed by returning only to step 5 of protocol B.
C. Large-Scale Preparation of Plasmid DNA:
The bacteria are harvested by centrifugation and then lysed in buffer solution containing lysozyme, to break open the bacterial cells walls, and the detergent SDS, to separate out all the cell contents. Addition of potassium acetate results in the formation of a heavy white precipitate of bacterial genomic DNA entangled in the remnants of the bacterial cells.
This precipitate is spun out, leaving the plasmid DNA still in solution. Addition of isopropanol to the supernatant then precipitates out the plasmid DNA, which is pelleted by centrifugation.
Unfortunately, these precipitations are not absolute and the plasmid DNA has to be purified from cell RNA and remnants of single-stranded bacterial DNA by equilibrium- density-gradient centrifugation in caesium chloride in the presence of saturating quantities of ethidium bromide.
Under these conditions, plasmid DNA bands at a higher density than bacterial cell RNA and single-stranded DNA remnants. Usually there are two bands of plasmid DNA about half-way down the gradient. The lower band is the supercoiled plasmid DNA.
The upper band is the open circular form of plasmid DNA and should be present in only low amounts in a good plasmid preparation. The lower supercoiled band of plasmid DNA is isolated, ethidium bromide is removed and the DNA is precipitated by addition of ethanol.
The plasmid DNA can finally be spun down as a pellet and re-dissolved in buffer for use. Quantities given in the following recipes are for preparation of plasmid from a 400 ml bacterial culture. If greater quantities are required the amounts given must be scaled up appropriately.
Equipments recommended:
Balance for weighing
Refrigerated Sorvall centrifuge at 4°C with rotor GSA (or equivalent)
250 ml plastic bottles for centrifuge
Bucket of ice
100 ml measuring cylinder
Glass funnel with muslin filter
10 ml polycarbonate centrifuge tube — Oak Ridge type
Beckman ultracentrifuge with rotor 50Ti (or equivalent)
30 ml Corex glass tubes (autoclaved)
Ultraviolet light
Glass Pasteur pipettes (autoclaved)
-20°C freezer
Spectrophotometer
Solutions and chemicals required:
Lysozyme (Sigma)
Solution I made as:
4.5 g D-glucose
12.5 ml 1 M Tris-HCl pH 8.0
10 ml 0.5 M EDTA
Made up to 500 ml with distilled water
Autoclave
Solution II made as:
20 ml 1 M NaOH (autoclaved)
10 ml 10% w/v SDS (Serva) in water (filter-sterilized 0.2 µm membrane)
70 ml distilled water (autoclaved)
This solution should be made freshly as required and, in any case, not stored for more than one week.
Solution III made as:
147.2 g potassium acetate
450 ml distilled water
Adjust pH to 5.0 with glacial acetic acid
Autoclave
10 × TE buffer made as:
40 ml 1 M Tris-HCl pH 8.0
8 ml 0.5 M EDTA
Make volume up to 400 ml with distilled water
Autoclave
Isopropanol (propan-2-ol)
Ethidium bromide solution 5 mg/ml in water
Caesium chloride
Iso-butanol
100% ethanol
70% v/v ethanol in water
Distilled water (autoclaved)
Procedure:
1. Pour the bacterial culture into 250 ml plastic bottles for the centrifuge.
2. Harvest the bacteria by centrifugation at 8,000 rpm (10,400 g) for 5 minutes in the Sorvall centrifuge using GSA rotor at 4°C.
3. Pour off the supernatant and drain the pellet.
4. Re-suspend the pellet from a 400 ml culture in 20 ml of solution I in a 250 ml plastic centrifuge bottle.
5. Add solid lysozyme to a final concentration of 5 mg/ml and mix to dissolve.
6. Keep at room temperature for 10 minutes.
7. Add 40 ml of solution II and mix well. The solution should turn very viscous at this stage.
8. Keep on ice for 5 minutes.
9. Add 20 ml of solution III and mix well. A heavy white precipitate should form.
10. Keep on ice for 15 minutes.
11. Spin down the precipitate at 8,000 rpm (10 400 g) for 5 minutes in the Sorvall centrifuge using GSA rotor at 4°C.
12. Take the supernatant. Pour it into a measuring cylinder through a muslin filter in a glass funnel to remove any lumps of precipitate. Measure the volume and transfer to a clean 250 ml plastic centrifuge bottle.
13. Add 0.6 volumes of isopropanol. A fine precipitate of plasmid DNA should now form.
14. Spin down the precipitate at 8,000 rpm (10,400 g) for 10 minutes in the Sorvall centrifuge using GSA rotor at 4°C.
15. Discard the supernatant by pouring off. Drain and dry the pellet, either for a few minutes in air or in a vacuum desiccator, if available.
16. Re-dissolve the precipitate in 5.4 ml of 10 x TE buffer and transfer to a Corex glass tube.
17. Add 0.6 ml of ethidium bromide (5 mg/ml) solution.
18. Add 6.1 g of solid caesium chloride.
19. Transfer to a 10 ml polycarbonate tube (Oak Ridge type).
20. At this stage, all tubes should be balanced to within 0.1 g, ready for centrifugation and tubes should be capped. Tubes should also be filled to within 1 ml of full capacity or else they can collapse during centrifugation — this can be done by overlaying with liquid paraffin, if necessary.
21. Centrifuge at 35,000 rpm for at least 40 hours (or, for convenience, over the weekend) at 15°C in a Beckman ultracentrifuge in a 50Ti rotor (111 000 – 51 300 g).
22. Remove tubes from the centrifuge and view in ultraviolet light. The plasmid DNA should be clearly visible as a single red band of supercoiled form in the middle of the gradient, although a second minor band of open circular plasmid is sometimes also obtained just above. The heavy red pellet at the base of the tube is of bacterial RNA and single-stranded DNA.
23. Collect the band of supercoiled plasmid DNA using a Pasteur pipette and transfer to a 30 ml Corex glass tube.
24. Add an equal volume of iso-butanol and vortex. Allow to stand for one minute to separate the phases. Discard the upper (pink) iso-butanol phase.
25. Repeat step 24 twice more until all ethidium bromide has been removed from the plasmid DNA solution.
26. Dilute the plasmid DNA solution 3 to 4 fold with distilled water and transfer to a clean Cortex glass tube. Measure the volume and add 2 volumes of 100% ethanol.
27. Keep at -20°C for 2 hours. The mixture must not be stored for any prolonged period or the caesium chloride will come out of solution.
28. Spin down the DNA precipitate at 10,000 rpm (12,000 g) at 4°C for 20 minutes in the Sorvall centrifuge (rotor SS34) or equivalent.
29. Wash the precipitate once with 70% ethanol and repeat step 28.
30. Drain and dry the DNA pellet for a few minutes at room temperature and re-dissolve it in 0.5 ml of distilled water.
31. The concentration of the DNA can be measured using optical density:
Take 10 µl of plasmid DNA solution.
Add 490 µl of distilled water.
Read optical density at 260 nm (OD260).
A solution of DNA in water of 1 mg/ml gives an OD260 of 20.
Thus µl/µl in DNA solution = 1/20 × OD260 × 50.
32. Store at -20°C.
D. Isolation of Cloned Gene Sequences:
The final steps involve purification of the cloned gene sequences from the plasmid. The plasmid DNA is cut with the appropriate restriction enzyme(s) used for the original gene cloning. This releases the cloned gene from the plasmid and the two DNAs can be separated by agarose gel electrophoresis.
Nuclease contamination must be avoided in procedures 1 and 3. For these protocols, gloves must be worn throughout; pipette tips, glassware and solutions should all be autoclaved before use. These precautions are not necessary during the running of the agarose gel (procedure 2).
Solutions and chemicals required:
10 × restriction enzyme (10 × RE) buffer made as:
2 ml 1 M Tris-HCl pH 7.4 (autoclaved)
1.6 ml distilled water (autoclaved)
0.4 ml M magnesium chloride (autoclaved)
28 µl stock p-mercaptoethanol
This can be stored at -20°C up to one month
Restriction enzyme preparation (Boehringer or Bio labs)
5 M sodium chloride (autoclaved)
Distilled water (autoclaved)
5 × TBE buffer made as:
106 g Tris base
55 g Boric acid
9.3 g di-potassium EDTA
Dissolve and make up to 2 litres with distilled water
1 × TBE buffer made by dilution of 5 × TBE buffer
Ethidium bromide (BDH) 10 mg/ml in water (store in the dark)
Agarose powder (SEAKEM-ME) (FMC Corp.)
DNA sample buffer (DNA SB) made as:
330 µl glycerol
75 µl 5 × TBE buffer
250 µl 0.5 M EDTA (pH 7.0)
Mix thoroughly. Then
Add 27 µl of 10% w/v SDS (Serva) in water (if this is added earlier it precipitates out)
Add bromophenol-blue powder (spatula tip, approx.100 µg)
Phenol, redistilled and water-saturated
(Rathburn Chemicals)
Chloroform
3 M sodium acetate pH 5.2
100% ethanol
70% v/v ethanol in water
Equipments recommended:
Micro centrifuge tubes 1.5 ml volume (autoclaved) (Treff Lab)
Water-bath at 37°C
Microwave oven or boiling water-bath
Agarose gel electrophoresis equipment
(commercially available or home-made)
Filter-paper wicks Electrophoresis power supply
Ultraviolet light Scalpel
Micro centrifuge (Eppendorf or M.S.E.)
Freezer at -20°C
Procedure:
1. The plasmid DNA is cut with the appropriate restriction enzyme(s) used for the original gene cloning, as:
(i) Place 20 µg of plasmid DNA in a 1.5 ml micro centrifuge tube,
(ii) Add 5 µl of 10 × RE buffer,
(iii) Add the required amount of enzyme (up to 5 µl volume only). The amount of enzyme required is calculated in units (1 unit enzyme cuts 1 µg of DNA in 1 hour at 37°C in a volume of 50 µl).
(iv) Add 5 M NaCl as needed by the enzyme.
There are three groups of enzymes:
Low-salt enzymes — add no 5 M NaCl
Medium-salt enzymes — add 0.5 µl of 5 M NaCl
High-salt enzymes — add 1.0 µl of 5 M NaCl
The salt requirements are specified by the supplier.
(v) Add distilled water to make the volume up to 50 µl.
(vi) Incubate reaction mixture for 1 hour minimum or, if convenient, overnight. Most enzymes work best at 37°C but some require other temperatures. This will be specified by the supplier.
2. The cloned gene is separated from plasmid sequences on a 0.8% agarose gel, as:
(i) Prepare mould for the gel. Many systems are available commercially for pouring gels but a simple home-made system is illustrated in Fig. 1.8:
A Perspex plate is levelled using a spirit level.
A glass plate (14 cm × 19 cm) is used as the mould. Its edges are sealed with PVC electrical tape (or radioactive/biohazard-labelled tape) so that it forms a wall 1 cm high all around the plate. This is placed on the level Perspex plate. A simple Perspex comb is assembled at one end of the plate so that there is about 1 mm of space between the base of the teeth and the glass plate.
(ii) Weigh 0.8 g of agarose into a 200 ml conical flask. .
Add 100 ml of 1 × TBE buffer.
Add 100 of ethidium bromide solution (10 mg/ml in water).
Heat in microwave oven or boiling water-bath until agarose dissolves.
(iii) Pour agarose onto gel mould and allow to set for 1-2 hours.
N.B.:
The recipe given here provides a 0.8% agarose gel, which is useful for most purpose. To separate the very large or very small DNA fragments, the concentration of agarose can be varied.
(For further details see Maniatis et al., 1982.)
(iv) The gel tank is prepared and connected to an electrophoresis power supply. Many systems are available commercially but a simple home-made system is illustrated in Fig. 1.9.
(v) Place 1,600 ml of 1 × TBE buffer into the gel tank and add 2 drops of ethidium bromide solution.
(vi) Put the gel into the tank. The surface of the gel should be just submerged (by about 1 cm depth of buffer).
(vii) To the 50 µl enzyme reaction mixture, add 20 µl of DNA SB.
(viii) Load the DNA into wells of the gel.
(ix) Load also some polynucleotides of known size in an adjacent well to provide molecular weight-markers. Common markers used are phage X DNA cut with the restriction enzyme Hind III (Bio labs). (See Table 3.1.)
(x) Run the gel for 2-4 hours at 120 v. All samples are run from the negative to the positive electrode.
3. The cloned gene is eluted from the gel as:
(i) View gel under ultraviolet light to locate the DNA bands. Cut, with a scalpel blade, a small trough right through the gel immediately in front of the band to be eluted (on the positive electrode side of the band). The trough should be the length of the DNA bank and about 3-5 mm wide.
(ii) Replace the gel onto a clean dry glass plate. Trickle some liquid agarose (0.8%) in 1 × TBE buffer as at step 2(ii) around the base of the trough to seal it. Allow to set for 5 minutes.
(iii) Fill the well with 1 × TBE buffer.
(iv) Place the gel back into the gel tank but with only 200 ml of 1 × TBE buffer, so that the gel is not covered with any buffer.
(v) Soak two pieces of filter paper in 1 × TBE buffer and assemble them as wicks from the gel to the buffer tank. This assembly is illustrated in Fig. 1.10.
(vi) Subject to electrophoresis at 500 V for 30 second bursts. View the gel after each burst to determine exactly where the DNA band is. When the DNA has moved into the buffer in the well, transfer it to a 1.5 ml micro centrifuge tube. If the DNA moves too far at any stage, the current can be reversed until the DNA is back in the well.
4. The eluted DNA is finally purified as:
(i) To the DNA solution in the micro centrifuge tube, add an equal volume of phenol.
(ii) Mix by vortexing.
(iii) Break the phases by centrifugation for 5 minutes in a micro centrifuge.
(iv) Transfer the upper aqueous layer to a clean 1.5 ml micro centrifuge tube.
ADVERTISEMENTS:
(v) Repeat steps 4(i)-4(iv) once.
(vi) Add an equal volume of chloroform.
(vii) Mix by vortexing.
(viii) Break the phases by centrifugation for 5 minutes in a micro centrifuge.
(ix) Transfer the upper aqueous layer to a clean 1.5 ml micro centrifuge tube.
(x) Add 1/10 x volume of 3 M sodium acetate pH 5.2 and mix.
(xi) Add 2 volumes of 100% ethanol.
(xii) Store at – 20°C overnight (about 18 hours). If the DNA is required urgently, the tube can be placed in dry ice and the DNA is then recoverable after 30 minutes.
(xiii) Spin down the DNA precipitate in a micro centrifuge for 15 minutes.
(xiv) Pour of the ethanol.
(xv) Wash the DNA pellet in 70% ethanol.
(xvi) Spin down the DNA precipitate in a micro centrifuge for 10 minutes.
(xvii) Pour off the ethanol. Drain and dry the DNA pellet for a few minutes.
(xviii) Re-dissolve the DNA in 20 ml of sterile distilled water.
(xix) Store at – 20°C and use in the nick translation reaction as required.
