C hapter 2 : Materials and Methods
2.8. DNA methods 1 Transformation
Transformation of bacteria with plasmid DNA containing cDNA fragments is essential if it is required to propagate many copies of a desired cDNA fragment. The use of competent bacteria enhances the uptake of plasmid DNA. The bacteria are made competent by treatment in an ice-cold environment with CaCl2 as described
by Maniatis et al ( 1982 ). The CaCl2 treatment alters the structure of the cell wall
and allows the plasmid DNA to enter into the cell.although this process varies markedly with strain and species of bacteria.
Two strains of competent bacteria ( INVF 1-a Escherichia coli, DS9AlEscherichia coli, Invitrogen, Leek, Neteherlands ) were used for transformation. Two strains were used as the efficiency of mansformation varied markedly with the size of the plasmid DNA containing the cDNA fragment being introduced. INVF 1-a
Escherichia coli proved to be excellent for sizes up to 6Kb however with larger
plasmid sizes the successful transformation was not achieved. For larger plasmid DNAs, DS941 Escherchia coli proved to be very successful yielding large numbers of transformants. The competised bacteria were stored in 200ql aliquots containing KCl lOOmM, K acetate lOmM, MnCl2, lOmM CaCl2, pH 6.2.0 with 20% glycerol
w / V to facilitate viability of the cells during the freezing process.
The plasmid containing the cDNA of interest ( Chapter 4 ) was diluted to 3 different concentrations 10, 50 and lOOng in lOjil of TE buffer, pH 8.0. The volume of the DNA should not exceed 5% of the volume of the transformation mixture as this impairs transfoimation efficiency ( Maniatis et al, 1982 ). The bacteria ( 200ql ) were defrosted on ice for 20 minutes. The plasmid DNA was mixed with the bacteria and left for a further 20-30 minutes on ice. The mixture was heat shocked for 1-2 minutes in a water bath at 42°C followed by immediate ti'ansfer to ice to quench the heat for 1-2 minutes to introduce the plasmid DNA into the cells. An aliquot ( 450|il ) of SOC medium ( tryptone 2%, 0.5% yeast extract, lOmM NaCl, 2.5mM K C l, lOmM MgCl2, lOmM MgSOz). and 20mM glucose ) was added to the
transformation mixture and incubated on a rotating wheel for 1 hour at 37 °C. Luria- Bertani agar ( tryptone 1%, yeast extract 0.5%, NaCl 1%, 1.5% agar ) was
prepared and autoclaved ( 120°C, 151bs / inch^, 15 minutes ), when the agar had cooled to 55°C, ampicillin was added to a final concentration of 50qg / ml and the agar poured into 90mm plates and allowed to set. Ampicillin is used as a selectable marker for recombinant clones as the plasmid DNA introduced into the cell contains the Amp^ gene which confers ampicillin resistance to recombinant cells. In order to further facilitate selection of recombinant clones a |3-galactosidase selection system was also used. This system allows the selection of clones which have the plasmid DNA containing the cDNA fragment of interest in the correct orientation. The (3-gal gene in the plasmid lies downstream of the multiple cloning site ( MCS ), if a cDNA fragment is inserted into the MCS this causes a change in the reading frame so that the (3-gal gene is not functionally expressed. This may also occur if the insert sequence alters the (3-gal gene sequence. The substrate Bluo-gal ( Bromo-4-chloro- 3-indoyl-(3-D-galactoside, cat no.540-5519UA, Gibco-BRL, Paisley, UK ) is used in conjunction with IPTG ( isopropylthio-(3-galactoside, cat.no.540-5529UA, Gibco-BRL ) which is an inducer of p-galactosidase activity, in the presence of a functional p-gal a blue indoyl derivative is produced. This selects for plasmids with functional P-gal activity which are not recombinants. Therefore recombinant clones containing the cDNA insert can be selected on the basis that they will be cleai’ ( disrupted p-gal gene ) and will not produce a blue colour.
A 2% Bluo-gal solution ( lOOql ) is spread plated to fomi a continuous layer on the top of the agar plates, additionally 50|il of lOOmM IPTG was spread onto the plate and allowed to dry. The ti'ansfomiation mixture was spread plated at 100|il / plate and incubated inverted at 37°C overnight. The recombinant clones containing ampicillin resistance and disrupted p-galactosidase activity could be easily identified as clear colonies and the false clones containing plasmid but no cDNA insert are blue.
2.8.2. Plasmid DNA mini-preparations
In order to positively identify that the correct clone has been obtained before scaling up to a lai'ge scale plasmid preparation, the authenticity of a recombinant clone can be quickly identified using a plasmid mini-preparation. The method of Maniatis et al
( 1982 ) was used with the following modifications.
Single colonies of recombinant clones were picked from the agar plates and
inoculated into 2ml of Luria-Bertani broth ( tryptone 1%, yeast extract 0.5%, NaCl 1% ) containing ampicillin ( 50jag/ml ) and incubated overnight at 37°C. The cells were harvested next day by centrifugation ( Biofuge, Heraus, Sepatech, Baujahr, Germany ) at 14926 g for 30 seconds at room temperature. Prior to cell harvesting 0.25 volumes of bacteria were sampled and glycerol added to 20% of the final volume. These cells were frozen at -20°C and used as innoculum after the clone of interest is identified.
The bacterial pellet obtained after centrifugation was re-suspended in 1 volume of Solution 1 ( 50mM glucose, 50mM Tris-HCl lOmM EDTA pH 8 ) and left on ice
for 5 minutes. Two volumes of Solution 2 ( 0.2N NaOH, 1% SDS ) were added and the tube inverted rapidly five times to ensure good mixing in the tube. Finally 1.5 volumes of Solution 3 ( 5M K acetate, 11.5% glacial acetic acid ) was added and the mixture vigorously mixed and stored on ice for 5 minutes. The resulting lysate was centrifuged at 14926 g for 30 seconds at 4 °C ( Biofuge, Heraus), and the supernatant collected. The supernatant obtained was extracted twice with an equal volume of phenol : chloroform ; iso-amyl alcohol ( 1 : 1 : 24 ) and once with an equal volume of chloroform : iso-amyl alcohol ( 1 : 24 ). The plasmid DNA in the extracted solution was precipitated by the addition of 2 volumes of absolute
alcohol at room temperature and collected by centrifugation at 14926 g for 15
minutes at room temperature. The DNA pellet obtained was washed in 1 volume of 70% ethanol and collected by centrifugation as before. The pellet was air dried and re-suspended in an appropriate volume of TE buffer ( lOmM Tris-HCl, ImM EDTA, pH 7.4 ) and stored at -20°C for further use.
Large scale plasmid preparations use essentially the same technique as described above with the volumes increased by a factor of 10. Cultures ( 500ml ) which were grown overnight after being seeded with the recombinant clone were used. In the large scale preparation iso-propanol was used to precipitate the plasmid DNA.
2.8.3. Plasmid DNA Isolation
A caesium chloride ( CsCl ) gradient was used to purify plasmid from the DNA extract. For each tube used in the gradient, 4.065g of CsCl was added to 3.815ml of TE buffer ( lOmM Tris-HCl, ImM EDTA, pH 7.4 ) containing the dissolved DNA extract and dissolved. After the CsCl had dissolved 120|U,1 of ethidium bromide ( lOmg/ml ) was mixed into the solution and tubes ( ultia-cleai",
13x51mm, Beckmann, High Wycombe, Bucks, UK ) were layered to the top with mineral oil. The density of the final solution was 1.6g / ml. The solution was centrifuged at 36,000rpm ( 116,000g ) at 20°C for >36 hours ( SW 55 Ti rotor, Beckmann L7 ultracentrifuge ). After centrifugation two red bands ( Ethidium bromide stain ) were located lying centrally in the tube, the upper band being 'nicked' ckcular plasmid DNA and the lower band closed circular supercoiled plasmid DNA. The plasmid DNA from both bands was recovered using a pasteur pipette inserted through the oil layer of the tube. The recovered solution was
extracted with an equal volume of water saturated butan-l-ol to remove the ethidium bromide and this was repeated until all trace of colour was removed from the
solution. The plasmid DNA was recovered by precipitation in 2 volumes of absolute ethanol and 0.1 volume of 3M Na acetate, pH 5.2, at -20°C overnight followed by centrifugation at 10,000rpm ( 15,700g ) for 30 minutes at 4°C ( JS13.1 rotor, Beckmann J2-MC high speed centrifuge ). The pellet was washed in 70% ethanol by centrifugation at 15,700g, 4°C for 30 minutes, air dried and re suspended in an appropriate volume of TE buffer.
2.8.4. Quantification of DNA
DNA was quantified spectrophotometrically following that described by Maniatis et al ( 1982 ). The quantity of DNA can be determined from the absorbance at 260nm of the DNA in solution ( using a path length of 1 cm ). One optical density unit is equal to a DNA concentration of 50p.g / ml. Using an optical density reading at 280nm the relative purity of the DNA can also be assessed. As protein
contamination in the solution would increase the relative absorbance at this
wavelength. A ratio obtained by dividing the 260nm reading by the 280nm reading gives an indirect measine of DNA purity. A protein-free DNA solution would have a 260 / 280 value of 2 and variation either side of this value suggests contamination
of the solution.
2.8.5. DNA restriction enzvme analvsis
DNA manipulations were performed using restriction enzyme digestion to cut the experimental cDNA at the required nucleotide sequence. A variety of restriction endonucleases were employed, most were obtained from Gibco / BRL, New England Biolabs, Hitchin, Staffs, UK and Northumbria Biologicals Ltd.,
Cramlington, Northumberland, UK. Restriction digests were performed either at 37°C or at room temperature for 3-16 hours in the salt buffers recommended by the manufacturers. The salt buffers used contained different concentrations of NaCl, KCl, Tris-HCl and Mg2Cl and have different pH values. The buffer used was
dependant on the optimal conditions required for the maximum activity of the specific restriction enzymes used. The units of enzyme activity are defined as the number.of units of activity required to fully digest substrate /i-DNA or a
comparable DNA substrate. The number of enzyme units required was calculated by dividing the number of specific sites for the required enzyme in the experimental DNA by the number of sites in X-DNA, this value was then multiplied against the actual enzyme units required to fully digest the X.-DNA in 1 hour at 37°C. Activity required for overnight digests was taken from guideline's set by the manufacturers, this relates to the number of enzyme units of activity surviving during a 16 hour ( overnight ) digest. This allowed the calculation of the minimum number of enzyme units required to complete an overnight digestion.
2.8.6. Agarose gel elecü'ophoresis
Agarose gel electrophoresis was employed to identify different DNAs and DNA fragments produced from restriction digests. A 1% w / v agarose gel ( Ultra-pure agarose, Gibco / BRL ) made up in a xl TAE buffer ( 40mM Tris-acetate, ImM EDTA, pH8.0 ) was used to seperate fragment sizes > 500bp. In preparation, the molten agarose solution was degassed in a vacuum oven to remove any air bubbles in the solution. Either 100ml or 30ml gel casts were used and the appropriate well comb which deteimines the number and volume of wells available was used dependent on the number of samples and purpose required. The gel was allowed to set for approximately 1 hour and after this time period the comb was removed and
the gel set into the electrophoresis tank. Electrophoresis tanks were obtained from both Scotlab ( Scotlab Ltd., Coatbridge, Scotland ) ( midi gel ) and Gibco / BRL ( mini gel ) and power supply units ( Pharmacia, model EPS 500 / 400, Milton Keynes, UK ). TAE buffer was added to a level approximately 50mm above the gel surface and the well comb removed. DNA samples were prepared in a loading buffer containing xlTAE, 0.2% w/v bromophenol blue, 0.2% w/v Xylene cyanol, 30% glycerol and loaded into the wells ( 30-50|al ). Standards used were X phage DNA digested with Hindi 11 ( 23-2.0 Kb, Gibco / BRL ) and (|) HAE 174
bacteriophage DNA digested with Hael 11 endonuclease ( 1.36-0.072 Kb, Gibco / BRL ). Samples were run in to the gel at 100 V and electiophoresed at no greater than 5V / cm. The electrophoresis was stopped after the bromophenol blue had migrated approximately 80% of the gel length dependant on the purpose of the gel. Staining of the seperated DNA in the agarose gel with ethidium bromide was carried out prior to electrophoresis by adding the ethidium bromide ( 0.5-Iqg / ml ) into the gel before casting. Ethidium bromide intercalates between the bases of the DNA and is fluorscent therefore can be visualized using a ultra-violet ( 302nm wavelength ) transilluminator ( UVP Inc., San Gabriel, USA ), the DNA bands, stained with ethidium bromide can then be recorded and photographed.
2.8.7. Recoverv of DNA from gels
Two methods were used to purify DNA ( 800-1350bp ) from agarose gels ; l.electroelution ( IB I electroeluter, International Biotechnologies Inc., New
Haven, CT, USA ) and 2.a Qiaex gel extraction kit ( Qiagen Inc., Chatsworth, CT, USA).
1.The electroelution technique involved electrophoresing the DNA band contained in a gel slice in a xO.5 TAE buffer. The gel slice containing the DNA is placed in a horseshoe shaped slot with a current applied the DNA moves into a V-shaped channel. The channel contains a small volume ( 75|il ) of high salt ( 3M Na acetate, pH 5.2 ) and 0,2% bromophenol blue to allow visual identification of the salt trap. The DNA loses mobility as the charge is transferred to the acetate which is in ionic excess and is effectively 'trapped'. The DNA was collected from the salt trap by precipitation in 2 volumes of absolute ethanol. The DNA was recovered by
centrifugation at 14926 g, for 15 minutes at room temperature ( Biofuge, Heraus ), washed in 70% ethanol, precipitated and then dissolved in TE buffer or DEP water to the required volume for direct use or in ethanol for storage at -70°C.
2.Recovery of DNA using the Qiaquick gel extraction kit ( Qiagen ) relies upon the adsorption of DNAs to silica gel beads in a chaotropic buffer. The gel slice and 3 volumes of chaotropic buffer ( QXl buffer, Qiagen ) were melted in a dry block at 50°C for 10 minutes and the solution homogenised. To the solution 1 volume of iso-propanol was added. The solution was applied to a column containing the silica beads and centrifuged at 14926 g, for 1 minute at room temperature ( Biofuge, Heraus. ). The DNA fragments bind to the beads and the chaotropic buffer, iso propanol and residual agarose are washed out of the column by the centrifugation. The column was washed twice through by centrifugation after the addition of an ethanol solution ( PE buffer, Qiagen ). DNA was eluted from the column by the addition of 50ql of Tris-HCl, pH 8.5 and centrifugation at 14926 g, for 1 minute at room temperature
Although these different methods were used for the recovery of DNA from agarose gels neither of the techniques appeared to yield a significantly greater proportion of DNA from the gel slice.
2.8.8. Radiolabelling of DNA
Complementary DNA probes ( 25ng ) were radiolabelled using a random primer plus extension labelling system kit containing a 14-mer random primer ( NEP-
112L, NEN Dupont.) The label ( 5\xl ) used was dCTP ( 3000 Ci / mmol, NEN Dupont ), which was incorporated into cDNAs synthesized from the template cDNA by incubation with ôjal dNTP mix minus dCTP ( 20mM GTP, 20mM ATP, 20mM TTP
),
6|il reaction buffer(
14-mer oligonucleotides, Tris-HCl, MgCl2,2-
mercaptoethanol, bovine serum albumin, pH 7.6 ), 2 jj.1 of the large fragment DNA polymerase 1 ( 1.5-2.5 units / p.1 ) in a buffer containing ( Tris-HCl, 50% glycerol, 2-mercaptoethanol, pH 7.5 ) and made to a final reaction volume of 30p.l with DEP water. After incubation for 30 minutes at 37°C the mixture was seperated by gravity on a 2ml Sephadex ( G50 ) column equilibrated in TE buffer pH 7.5. The mixture was added to the column and the progress of the radiolabel was monitored through the column using a geiger counter ( mini-1, series 900, Mini instruments Ltd, Burnham on Crouch, Essex, UK). As the radiolabel reached the bottom of the column the eluate was collected into 1.5 ml microfuge tubes at 4 drops / tube. A total of 10 tubes were collected, from each tube a fraction ( I p l ) was sampled into
3ml of water and radioactivity was detennined on a scintillation counter ( 1600 TR liquid scintillation counter, Packard ) by cerenkov counting. The labelled cDNA was eluted in the first 4 fractions as the radiolabelled cDNA passes between the sephadex beads as it is excluded from entering the beads which have a small pore size. A second peak was obseived in factions 7-9, this was the non-incorporated a^2p dCTP which enters the dextran beads and therefore has a longer pathlength to the bottom of the column. The percentage incorporation of the label and the specific activity of the cDNA can be calculated from these readings.
End labelling utilizes the enzyme T4 poly nucleotide kinase ( T4 PNK ). This enzyme catalyses an exchange of a phosphate which in this case was y-32p ATP ( 6000 Ci / mmol, NEN Dupont ) with the 5'phosphate of the DNA molecule. The y- 32p ATP ( 5 p.1 ) was incubated with 5 \x\ PNK reaction buffer ( 250mM Imidiazole- HCl, 60mM MgCl2, 5mM 2-mercaptoethanol, 350qM ADP, pH 6.4 ), lOjil of 18S
ssDNA ( 3 0 n g / p i ) , 2pl T4 poly nucleotide kinase ( 10 units / pi ),300ng of single stranded oligonucleotide nucleotide and DEP water added to a final volume of 25pi for 1 hour at 37°C. The reaction products were seperated as detailed in the above section using a 2 ml Sephadex ( G25 ) column.