Formulación matemática

water. The solution was autoclaved and stored at 4"C until use.

Tris EDTA (TE) buffer (pH 8.0): lOmM tris.Cl (pH 8.0), ImM EDTA (pH 8.0). The solution was aliquotted, autoclaved and stored at room temperature until use.

2.4.1 Small scale preparation of plasmid DNA.

This procedure is based on that described in Sambrook et al. (1989) using alkaline lysis of the bacteria. 10ml LB medium containing the relevant antibiotic was inoculated either with a single colony picked from an agar plate using a sterile platinum wire loop or by using a sterile platinum wire loop to scrape the surface of a frozen glycerol stock before swirling it in the medium. The culture was incubated at 37 °C overnight with vigorous

shaking. 1.5ml aliquots of the culture were pipetted into sterile eppendorf tubes and centrifuged in an eppendorf centrifuge for 10 minutes. The supernatant was decanted leaving the pellet as dry as possible. The pellet w as resuspended in lOOjil ice cold solution 1 and incubated at room temperature for 5 minutes. 200pl solution 2 were added, mixed by shaking, and the tube was stored on ice for 5 minutes. ISOpl of ice cold solution 3 were added, the tube was gently vortexed and incubated on ice for 5 minutes. The mixture was centrifuged for 5 minutes in an eppendorf centrifuge and the supernatant was decanted into a fresh tube. An equal volume of 1 : 1 phenol and chloroform (pH 8.0) was added and the tube vortexed. The mixture was centrifuged for 2 minutes in an eppendorf centrifuge. The aqueous layer was transferred to a fresh tube. Two volumes of 100% ethanol were added and mixed by inversion. The tube was kept at room temperature for 5 minutes and then centrifuged in an eppendorf centrifuge for 10 minutes. The supernatant was removed by aspiration. The pellet was washed with 70% ethanol, vortexed briefly and recentrifuged. The ethanol was drawn off as fully as possible and the pellet was dried under vacuum. The pellet was resuspended in lOpl TE buffer and stored at -20 “C until use. The DNA present was analysed by restriction digest (section 2.6).

2.4.2 M edium scale plasmid preparation.

This procedure is based on that described in Sambrook et al. (1989) using alkaline lysis of the bacteria. LB medium (100ml) containing the appropriate antibiotic was inoculated with the bacteria to be cultured. The culture was incubated at 3 7 'C overnight with vigorous shaking. The culture was poured into sterile plastic centrifuge bottles and centrifuged at 6000g, 4°C, for 10 minutes in a Sorval fixed angle rotor. The supernatant was discarded and

each pellet was fully resuspended in 4ml ice cold solution 1 by pipetting. The suspension was transferred to sterile 30ml plastic centrifuge tubes kept at room temperature for 5 minutes. 8ml of solution 2 were added and the mixture shaken well, then left on ice for 10 minutes. 4ml of ice cold solu tion 3 were added, the tube shaken and incubated on ice for 10 minutes. The tubes were centrifuged at 30 OOOg, 4°C, for 30 minutes. The supernatant was decanted through gauze into 30ml corex tubes and 0.6 volum es of isopropanol were added. The solution was mixed by inversion and left at room temperature for 15 minutes. The corex tubes were fitted with rubber adapters and centrifuged at 15 OOOg, room temperature, for 30 minutes. The supernatant was discarded and the pellet washed briefly with 70% ethanol. The ethanol was removed as fully as possible by aspiration. The pellet was dried under vacuum, resuspended in 1.25ml TE buffer and transferred to a sterile eppendorf tube. 39jil 5M NaCl and 25pl boiled RNase A (lOmg/ml) were added and the tubes incubated at 37 °C for 60 minutes. The reaction was split between two tubes and an equivalent volume of phenol (pH 8.0) was added and the mixture vortexed thoroughly. The samples were centrifuged for 5 minutes in an eppendorf centrifuge and the aqueous layer was transferred to a fresh tube. The extraction was repeated with 1 :1 phenol and chloroform (pH 8.0) and then with chloroform alone. The aqueous phase was mixed with 2 volumes of 100% ethanol and 0.1 volumes of 3M NaOAc and stored at -20 °C for at least an hour and preferably overnight. The samples were centrifuged for 10 minutes in an eppendorf centrifuge and the supernatant discarded. The pellet was washed with 70% ethanol and then dried under vacuum for 5 minutes. The pellet was resuspended in lOpl TE buffer. The solution was heated at 65 °C for 3 minutes and stored at -20 °C until use.

2.4.3 M edium scale plasmid D N A preparation using Oiagen columns.

Solutions:

PI solution: lOOpg/ml RNase A, 50mM tris.Q (pH 8.0), lOmM EDTA (pH 8.0). The solution was stored at 4°C until use.

P2 buffer: 200mM NaOH, 1% SDS. The solution was stored at room temperature until use.

P3 buffer: 3M potassium acetate (pH 5.5). The solution was stored at 4°C until use.

QBT buffer: 750mM N aQ , 50mM MOPS, 15% v / v ethanol, 0.15% Triton X- 100, pH 7.0. The solution was stored at room temperature until use.

QC buffer: IM N aQ , 50mM MOPS, 15% v / v ethanol, pH 7.0. The solution was stored at room temperature until required.

QF buffer: 1.25M N aQ , 50mM tris.Q, 15% v / v ethanol, pH 8.5. The solution was stored at room temperature until use.

Protocol:

LB medium (100ml culture per Qiagen Tip 500) containing the appropriate antibiotic was inoculated with the bacteria to be grown (section 2.1.3). The culture was incubated at 37 °C overnight, with vigorous shaking. The culture was poured into sterile plastic centrifuge bottles and centrifuged at 6000g, 4 “C, for 10-15 minutes. The supernatant was discarded and 10ml PI solution were used to fully resuspend the bacterial pellet. The mixture was transferred to 50ml plastic centrifuge tubes. 10ml P2 buffer were added, mixed by several inversions and kept at room temperature for 5 minutes. 10ml ice cold P3 buffer were added, mixed by inversion and incubated on ice for 20 minutes. The tubes were centrifuged at 30 OOOg, 4°C, for 30 minutes. In the meantime, the Qiagen Tip 500 was equilibrated with 10ml QBT buffer. After centrifugation, the supernatant was passed through sterile gauze into a

sterile 50ml plastic tube and poured onto the Qiagen Tip. The Qiagen Tip was washed twice with 30ml of QC buffer. A sterile glass corex tube was placed under the Qiagen Tip and 15ml QF buffer were added to the column. 10.5ml (0.7 volumes) of isopropanol were added to the corex tube. The tube was placed in a rubber adapter and centrifuged at 15 OOOg, 4°C, for 30 minutes. The supernatant was discarded and 15ml 70% ethanol used to wash the pellet. The tube was centrifuged at 15 OOOg for 15 minutes. The supernatant was discarded and the tube inverted on tissue to allow the remaining ethanol to drain away. When dry, the pellet was resuspended in 500jil TE buffer and stored at -20 “C until use.

2.5 Measurement of DNA and RNA concentration.

2.5.1 The optical density method.

The absorbance of an aqueous solution of DNA or RNA at X=260nm is proportional to the amount of DN A or RNA present (Sambrook et al., 1989). The optical density at X=260nm (A2 6 0) of the D N A or RNA solution diluted

in distilled water was measured using a Phillips PU 8720 U V /V is scanning spectrometer. The solution was contained in 1ml quartz cuvettes and the spectrophotometer was zeroed using a distilled water blank.

For double stranded DNA:

concentration (jig/m l) = 50 x A2 6 0 x dilution factor

For single stranded DNA:

concentration (pg/m l) = 37 \ A2 6 0 x dilution factor

For RNA:

2,52 Using DNA DipStick™ (Invitrogen).

This method was used to measure accurately low concentrations of DN A or RNA (0.1 to 10ng/|il). The kit was used according to the manufacturer's instructions.

2.6 Restriction digests.

Solutions:

lOx One-Phor-All buffer (Pharmacia): lOOmM tris.acetate (pH 7.5), lOOmM magnesium acetate and 500mM potassium acetate. This was used with Pharmacia enzymes according to the manufacturers instructions.