A NIVEL NACIONAL

90

according to the manufactures instructions. 5 of ligation reaction was

added to 200 \A of competent cells in pre-cooled 10 ml falcon tubes and left on ice for 40 minutes. The cells were then heat shocked for exactly 45 seconds in a water bath at 37°C and immediately placed back on ice for 2 minutes. The cells were then incubated with 1 ml of L-hroth for 1 hour to allow expression of the selective gene. A range of aliquots of cells were plated onto L-agar plates containing 100 pg/ml ampicillin, allowed to dry

and incubated overnight at 37°C.

2,3.2 P r e p a r a tio n o f p l a s m id DNA

Plasmid DNA was purified from E. coli using an alkaline lysis method as described by (Birnboim, and Doly, 1979) and modified by (Fehciello and Chinali, 1993).

Large scale isolation of plasm id DNA

Single bacterial colonies were picked from a selective plate into a 10 ml culture of L-broth containing the appropriate antibiotic and grown

overnight at 37°C with shaking. This culture was then used to inoculate IL of L-hroth with selective antibiotic and incubated as before. CeUs were then coUected by centrifugation at lOOOg. The pellet was washed with STE and resuspended thoroughly in 20 ml of ice cold solution I. To this was added 40 ml of freshly prepared solution II and the suspension mixed by

gentle inversion so as not to shear the chromosomal DNA. After 20

acid was added and mixed thoroughly. The resulting precipitate was

stored on ice for 20 m inutes. The mixture was then split into four 30 ml

sorvall centrifuge tubes and centrifuged at 12 OOOg for 15 m inutes at 0“C.

The resulting supernatant was precipitated with 0.5 volumes of

isopropanol and centrifuged for 15 m inutes at 12 OOOg. The precipitate

was then dried briefly, the pellets collected and resuspended in a total of

20 ml of TE buffer to which was added an equal volume of 4 M ammonium

acetate. The suspension was stored on ice for 20 m inutes then centrifuged

at 12 OOOg for 5 m inutes at 0°C. The supernatant was transferred to a

fresh tube and precipitated with 0.5 volumes of isopropanol and

centrifuged at 12 OOOg for 5 minutes. The pellet was then resuspended in

4 ml TE buffer containing 10 pg/ml of RNase (DNase free, Promega) and

left at room temperature for 20 minutes. To this mixture was added 4.8 ml

of 88% isopropanol-0.2 M potassium acetate. The mixture was then

vortexed and left at room temperature for 10 minutes. The sample was

then centrifuged for 5 m inutes at 12 OOOg and the supernatant rapidly

poured off. The tubes (without hds) were put back into the centrifuge at

their original orientation and centrifuged for 2-3 m inutes at 1 OOOg. The

la st traces of isopropanol were removed. The barely visible pellet was then

dissolved in 300 pi of TE and stored at -20°C.

Sm all scale p la sm id preparation

The above procedure was scaled down so that 10 ml cultures were

92

STE in a 2 ml centrifuge tube. This was then centrifuged for 1 minute at 6

OOOg and the supernatant fully aspirated off. The pellet was resuspended

in 250 pi of solution I and left on ice for 5 minutes, to this was added 500

pi of freshly prepared solution II and gently mixed. 750 pi of 4 M

potassium acetate-2M acetic acid was added to the supernatant and left on

ice for 5 minutes. The mixture was then precipitated by 0.7 ml of

isopropanol. The pellet was dissolved in 250 pi of TE/RNase buffer left for

15 m inutes and precipitated with 300pl of 88% isopropanol-0.2 M

ammonium acetate, vortexed and left for 10 m inutes at room temperature.

The supernatant was poured off (not aspirated) and the pellet dissolved in

100 pi of TE and stored at -20°C.

2.3.3 R e s tr ic tio n e n zy m e d ig e s tio n

Plasm id DNA was digested with restriction enzymes using the conditions

described by the manufacturer (Promega). Digestion was carried out with

10 units of enzyme for 1 pg of plasmid DNA at the recommended

temperature for between 1-3 hours.

2.3.4 P r e p a r a t i o n o f p r o b e D N A

Vector constructs containing cDNA prohes for northern analysis were

digested by restriction enzymes then electrophoresed on a low melting

point agarose gel. Under a UV transiUuminator, probe fragments were cut

manufactured recommendations (USB). The DNA was precipitated with

ethanol and resuspended in 500 pi of TE buffer.

2.3.5 P r e p a r a t i o n o f R N A f o r n o r th e r n a n a ly s is

M essenger RNA was used for northern analysis to m inimise non specific

binding of the probe to ribosomal RNA found in preparations of total RNA.

M essenger RNA from 50 x 10® cells was isolated using the dynabeads

mRNA DIRECT kit (DYNAL) as directed in the manufactures

instructions. In this method magnetic beads coated with oligo d(T) were

used to bind to the 3’ poly (A) regions of mRNA in cell lysates. The sample

was then subjected to a magnetic field and the magnetic bead/mRNA complex captured while the supernatant was aspirated off. The beads

were then washed in a TRIS buffer to remove the remaining cellular

debris and the mRNA released by washing in a low salt buffer. The RNA was then stored in at -70°C. In a typical three day experiment, the starting

50 X 10® E- ceUs yielded between 0.4 and 1 pg of RNA.

2.3.6 P r e p a r a t i o n o f R N A f o r R T P C R Preparation from lymphocytes

RNA was prepared from peripheral blood mononuclear cells (PBMCs),

EBV transformed B cell lines and the Jurkat T cell line. PBMCs were

separated from blood collected in sterile preservative-free heparin coated

tubes (less than 24 hours old) using FicoU (Pharmacia) separation (Section

94

(approximately 5mls). The density of live cells was determined by trypan

blue exclusion, as described in Section 2.2.3. Approximately 10® viable

cells were resuspended in lOOpl of solution D. RNA was then prepared

from the mononuclear layer using acid guanidinium tbiocyanate-pbenol-

cbloroform extraction (Cbomczynski and Saccbi, 1987) and stored in

isopropanol at -70®C. Before use, the sample was centrifuged at 12,000g