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2.2.1.1 Preparation of genomic DNA from mouse liver or kidney.

Livers and kidneys were removed from mice and frozen in liquid nitrogen as quickly as possible. A slice of frozen liver or kidney was removed with a scalpel, and placed into 350|il TNE solution (see Appendix 2A.1). The tissue was homogenised using a sterile Treff homogeniser (Scotlab) until completely broken down. The cells were lysed by addition of 5 0 |li1 of 10% SDS. An equal volume of saturated phenol was added and the tube shaken vigorously for 1 minute. The phases were separated by centrifugation at

13,400g for 5 minutes, the bottom layer discarded, and the extraction repeated. An equal volume of ether was added and the extraction repeated except that the top layer was discarded. The DNA was precipitated hy addition of 0.1 x volume of 3M sodium acetate pH5.3, followed by 3 volumes of absolute ethanol. After inverting the tube several times, the DNA was removed immediately using a sterile plastic inoculation loop. The DNA was resuspended in an appropriate volume of 1 x TE (lOmM Tris HCl / ImM EDTA, pH8.0) and stored at 4°C.

2.2.1.::. Large scale preparation (maxiprep) of plasmid and cosmid DNA.

Solution I 50mM glucose

25mM Tris HCl (pH8.0) lOmM Na%EDTA (pH8.0)

Solution II 0.2M sodium hydroxide 1%SDS

Solution III 5M potassium acetate glacial acetic acid H2O TOTAL 60ml 11.5ml 28.5ml 100ml

The resulting solution is 5M with respect to acetate and 3M with respect to potassium.

5ml of LB-broth (see appendix 2A-2), containing the appropriate antibiotics (appendix 2A-3), was inoculated with a single colony or 20|il of glycerol stock. This culture was incubated for 4-7 hours at 37°C with vigorous shaking. The 5ml culture was then used to inoculate two 250ml volumes of LB-broth in 1 litre flasks. These were incubated overnight at 37°C with vigorous shaking. After incubation the cells were pelleted by centrifugation at 9,000g for lOminutes. Each pellet from 250ml of culture was resuspended in 5ml of solution I, (i.e. 10ml in total) until a completely homogenous suspension was obtained. 20ml of freshly made solution II was added and the mixture was mixed by gentle swirling and cooled on ice for 10 minutes. 15ml of solution HI was added and the mixture shaken vigorously. The lysate was then spun at 10,000g for 20minutes at 4°C in 30ml Sarstedt polycarbonate tubes, and the supernatant removed (the debris sticks to the bottom of this particular type of tube). 0.6 x volumes of propan-2-ol was added to the supernatant, and the DNA recovered by centrifugation at 10,000g for 20minutes at room temperature. The DNA pellet was washed with 70% ethanol and dissolved in 11ml

10 X TE. After the DNA was dissolved, the following were added: 0.44ml 0.2M potassium phosphate pH8.0, 11.44g caesium chloride, and 1.14ml ethidium bromide solution (lOmg/ml). The DNA solution was then spun overnight at 45,000g at 20°C in a Sorvall Ultracentrifuge Vertical rotor. The following day the band of supercoiled double stranded DNA was located using UV illumination and removed from the tube by means of a syringe and needle. The resulting solution was extracted with an equal volume of propan-2-ol saturated with caesium chloride to remove the ethidium bromide. This procedure was repeated until the DNA solution was colourless, and then the solution dialysed against 1 x TE three times for Ihour to remove the caesium chloride. The DNA was precipitated by the addition of 0.1 x volume 3M sodium acetate pH5.3 and 3 volumes of absolute ethanol, and centrifugation at 10,000g for 10 minutes. The DNA was resuspended in an appropriate volume of 1 x TE (lOmM Tris HCl / ImM EDTA, pH8.0) and stored at -20°C. Recently the ultracentrifuge and following steps have been replaced by the use of the Wizard Magic Maxiprep purification system that utilises a purification resin which binds DNA. The procedure is much quicker and simpler but has not been used in the course of this project.

Small scale preparation (miniprep) of plasmid and cosmid DNA.

10ml of LB-broth, containing the appropriate antibiotics, was inoculated with a single colony or 20pl of glycerol stock. This culture was incubated overnight at 37°C with vigorous shaking. After incubation the cells were pelleted by centrifugation at 13,400g for 5minutes. Each pellet was resuspended in lOOpl of solution I, until a completely homogenous suspension was obtained. 200pl of freshly made solution II was added and the mixture was mixed by flicking the tube. 150pl of solution III was added and the mixture shaken vigorously. The lysate was then spun at 13,400g for lOminutes, and the supernatant removed. An equal volume of saturated phenol was added and the tube shaken vigorously for 1 minute. The phases were separated by centrifugation at 13,400g for 5 minutes, and the bottom layer discarded. The DNA was recovered by addition of 3 volumes of absolute ethanol and centrifugation at 13,400g for 20mins at room temperature. The supernatant was discarded and the DNA pellet washed with 70% ethanol. The pellet was dissolved in 50p,l TE and stored at -20°C. When this DNA was subjected to restriction enzyme digestion 2\x\ of DNase free RNase (Appendix 2A.1) was added to the reaction.