Nivel de Conocimiento Actual de los Usuarios Sobre

2 , 2 . 1 4 , 1 R a n d o m p r i m e d D N A h y b r i d i s a t i o n

2 .2 .1 4 . 1 . 1 Hybridisation

DNA b earin g nylon m em branes (in situ YAC, cosm id, cDNA or sou th ern b lo ts) w ere h y b rid ised according to Church and G ilb ert (1984). M em branes w ere p reh y b rid ise d in 'C h u rc h ' b u ffe r (0 .5 M so d iu m p h o s p h a te pH 7 .2 (note: 0 .5 M with respect to Na+), 7% (w /v) sod iu m dodecyl sulphate (SD S), 1 mM EDTA, 0.1 mg ml ^ yeast t-R N A ) in a sealed p lastic bag at 65°C for at least 30 min. Random prim e labelled DNA was heated to 100°C for 3 min. and then added to C hurch b u ffer to a final concentration o f 1 x 10^ cpm ml k P re h y b rid ise d m em branes were drained, h y b ridisatio n so lution added and h y b rid ised at 65°C for 3 hours to overnight.

2 . 2 . 1 4 . 1 . 2 Wash ing

H y b rid isatio n bags were unsealed the hy b rid isation solution drained o ff and the m em branes tra n sfe rred into 1 litre w ash so lution (40 mM so d iu m p h o s p h a te pH 7 .2 , 1% (w /v) SDS) at room tem perature for 5 min. M em branes were then tra n sfe rred into fresh w ash so lu tio n at room tem p eratu re and incubated at 6 5 °C fo r 20 min. If m em branes still con tained > 1 0 0 cps as m easured by a hand held G e ig er-m o n ito r then the la st w ash was repeated a second time. Membranes w ere b riefly air dried on W hatm an 3M filter p aper to rem ove excess liquid and then w rapped air tig h t in saran w rap and used for auto rad io g raph y .

2 . 2 . 1 4 . 1 . 3 Stripping

for 30 min. F ilters w ere dried betw een Whatman 3M filters and stored dry.

2 , 2 . 1 4 , 2 O l i g o n u c l e o t i d e h y b r i d i s a t i o n

2 .2 .1 4 .2 .1 Hybridisation

F o r oligo n u cleo tid e h y b rid isatio n only filters w ith cDNA PC R products w ere used. The nylon m em branes were briefly placed in SS arc b u ffer (60 0 mM so d iu m chloride, 60 mM sodium citrate, 7 .2 % (w /v ) sodium lauroly sarco sin ate (S ark o sy l N 30, BD H)) to p re-eq u ilibrate. Oligos w ere labelled at 5' ends with [^^P-y] ATP u sin g T4 po lyn u cleotid e kin ase and h yb rid ised at 4 nM in S S arc b u ffer, at 4°C for 3 hours - o vernight. Typically tw o 22 cm x 22 cm m em branes w ere h y b rid ise d in one 300 mm glass bottle with 30 mm diameter (H y b aid ) in a volum e o f 10 ml. F ilters were separated by tw o 23 cm x 23 cm nylon meshes (H ybaid) to allow free access to the h y b rid isatio n so lution to all areas o f the m em branes. Bottles were sealed with a ru b b e r b u ng and rotated h o rizontally on a p u rp o se built roller at 5 rpm.

2 .2 .1 4. 2 .2 W a $hing

A fter hy b rid isatio n m em branes were rin sed briefly in cold (4®C) S S arc b u ffe r and w ashed to g eth er w ith the nylon m eshes in 1 litre S S arc b u ffe r in a p o ly p ro p y len e lunch box at 10®C fo r 1 5 - 3 0 min. Up to 8 m em branes w ere w ashed to geth er in 1 litre S S arc even if they had been h y b rid ise d w ith d ifferen t oligo n u cleo tid es. A fter w a s h in g m em branes w ere separated from the nylon m eshes and drained o f excess liquid on W hatm an 3M paper at room tem perature. M embranes w ere never allow ed to dry com pletely w hile radioactive. M embranes w ere placed onto the p o ly th en e side o f B enchkote (W hatm an) and w rap p ed air tight in saran w rap and used for au to radio graph y .

2 . 2 . 1 4 . 2 . 3 S t r i p p in g

To rem ove all bound radioactive o ligonucleotide, up to 20 m embranes w ere incubated twice in 1 litre 0.1 x SSarc at 65®C for 10 min. The 0.1 X SS arc was boiled immediately prio r to p ouring onto the mem branes. After strip pin g m em branes w ere stored either in S S arc b u ffe r or used im m ediately in another h y brid isation .

2.2.15

DNA sequencing

S eq uencing o f cDNAs was perform ed by a did eox y -term in ation method on double strand DNA. All sequencing reactions w ere carried out using a S eq u enase v 2 .0 DNA sequencing kit (U B S , Cleveland, USA). DNA was prepared by alkaline lysis from 5 ml o f an o v ern ig h t culture and resu sp en d ed in 20 p.1 water. To denature the tem plate DNA, 5 p i o f a prep were diluted w ith 13 pi w ater and 2 pi o f 2 M NaOH, 2 mM EDTA w ere added. The DNA was precipitated with 2 p i 3 M NaAc pH6 and 50 p i ethanol (100% ). After cen trifu g atio n the pellet was w ashed once in 70% ethanol and resu sp en d ed in 7 p i water.

To anneal the sequencing prim er, 1 pi prim er and 2 p i sequencing b u ffer w ere added. The sam ple was then heated in a 65°C w aterbath and allow ed to cool to 35°C over a period o f 30 min. S eq u en cin g reactions w ere then carried out, u sing [3 5 S -a ]d A T P , as sh o w n in the protocol su p p lied with the kit.

The seq u en cin g reactions w ere run out on 30cm x 40 cm 6% p o ly acrylam ide den atu rin g gels (6 M urea) at 60 mA (c o n sta n t W atts) for 4 h o u rs.