7TCTTTTGGAGGTGCTCGGT

M odification: b io tin in corporation

G AM AS P ro m o ter CAACGAGCrCTACAAGGACrGGAGCCT GAMAS SST CCIGAGTGCAGCGCAGCICAGCCAG G S sense TCCITCGAAAGGCCACAGTC R V p rim e rS CTAGCAAAATAGGCTGTCCC 69

I shift GTGCrCGGTGTGCATGTG II sh ift CACATGCACAŒGAGCAC III sh ift GTIGCCrGATGCTTGCrr IV shift AGCAGATCTGGGTTAGCG PARA 50 CTGCTAAAAATCAATAAG PARA 50 Alt AACCCAGATCTGCTAAAA PARA 130 TCmTGGAGGTGCrCGGT PA RA 130 Alt GGGTTCmrGGAGGTGCr

2 .1 .9 Solutions and M edia

A garose gel loading buffer: 0 .2 5 % (w /v ) b r o m o p h e n o l b lu e, 0.25% (w /v) x y len e cyanol 25 mM E D TA , 50% (v/v) g ly c e ro l.

Phosphate buffered saline (PBS): 135 mM N aC l, 27 mM KCl, 10 mM Na2H P0 4, 15 mM KH2PO4 B

20x SSC: 3 M NaCl, 0.3 M trisodium citrate.

50x TAB: 2 M Tris, 50 mM EDTA pH 8.0

lOx TEE: 1 M Tris, 1 M boric acid, 20 mM EDTA pH 8.35

TE: 10 mM Tris-HCl, 1 mM EDTA pH8.0

L uria-B ertani (LB) m edia: 1% (w /v) b acto -try p to n e, 0.5% (w /v) bacto-yeast extract, 1% (w/v) NaCl.

LB for plates: 1.5% (w/v) agar in LB m edia

2 .2 M o l e c u l a r B i o l o g y T e c h n i q u e s

2 .2 .1 G e n e ra l

W henever possible, tubes w ere kept capped and on ice, to m inim ize evaporation o f the sm all volum es em ployed and lessen D N A dénaturation.

A d d itio n s w ere m ade w ith d isp o sa b le -tip m ic ro p ip e tte s and care was taken not to contam inate stock solutions.

The solutions w ere thoroughly m ixed after each addition, ty p ic ally by pum ping the solution tw o or th ree tim es w ith a m ic ro p ip e tte . C are w as tak en to av o id the c re a tio n o f air b u b b le s .

A t any stage w here it was possible for som e solution to clin g to the w alls o f the E p p en d o rf tu b es, the tubes w ere c e n tr if u g e d .

A ll the solutions, unless otherw ise stated, w ere m ade up w ith au to clav ed d o u b le-d istilled w ater.

2 .2 .2 C o nstruction o f reco m b in an t plasm ids

A ll rec o m b in a n t p lasm id s w ere co n stru cted acco rd in g to standard protocols (Sam brook et al, 1989). T ypically, 1-5 jig of the v ecto r and an eq u iv alen t am ount o f the in se rt D N A w ere d ig e s te d w ith the a p p ro p ria te re s tric tio n e n zy m e(s) in the rea ctio n b u ffer sp ecified and/or pro v id ed by the m an u factu rer.

The digests were incubated at 37° C for 2-4 hours, and checked by ru n n in g approxim ately 10% o f each reactio n on an agarose gel. If the d ig estio n was co m p lete, the rem ain in g D N A was separated on a TAB agarose gel and the desired bands p u rified u sin g p h en o l/ch lo ro fo rm e x tra ctio n (2 .2 .7 .1 ) and K ristal gelex (2 .2 .7 .3 ). S m all fra g m e n ts (< 1 0 0 bp) e x c ise d d u rin g the re stric tio n d ig estio n o f v ecto r D N A w ere rem oved during this p ro ced u re due to the in ab ility o f K ristal G elex to bind D N A fragm ents sm aller than 100 bp. D N A longer than 100 bp was se p ara ted from v ecto r D N A on a low m eltin g p o in t (L M P) agarose gel and the vector frag m en t was p u rified using K ristal G elex or phenol/chloroform .

W hen a single restriction enzym e was used, the vector was p h o sp h atased to p rev en t re c irc u la risa tio n by d ig estin g w ith 10

u n its o f c a lf in te stin al p h o sp h atase at 37°C for 30 m in u tes, f o llo w e d b y p h e n o l/c h lo r o f o r m e x tr a c tio n an d e th a n o l p r e c ip ita tio n .

V ecto r and in sert frag m en ts w ere resu sp e n d ed in sm all v o lu m e s o f a u to c la v e d d o u b le -d e io n is e d w a te r, and th e ir co n cen tratio n s estim ated by running a sm all aliquot o f each on an agarose gel next to known quantities of DNA m arkers.

A stan d ard lig atio n reactio n o f v ecto r and in se rt D N A w ould contain 100 ng o f vector, 10-100 ng o f insert preparation and 1 |LLl (10 units) of T4 DNA ligase, in 10 jLll o f reaction buffer sp e cified and p ro v id ed by the m an u factu rer. F or each set o f lig a tio n s , tw o or th ree re a c tio n s w ith d iffe re n t am ounts o f

vector and insert DNA were set up (usually 1:1, 1:2 and 1:10), ensuring that suitable m olar ratios o f the two w ere obtained. In p arallel, several control reactions w ere also set up to m o n ito r the efficiency o f ligation.

T he p ro d u cts o f the lig a tio n w ere m ix ed b rie fly and in cu b ated for 1-3 hours at room tem perature or 6-12 hours at 4°C . T h ey w ere tra n sfo rm e d in to c o m p e te n t b a c te ria (see 2 .2 .5 .1 ) w hich w ere su b seq u en tly p lated on to L B -ag ar (See

2.2.5.2) containing the appropriate antibiotic for selection.

T ypically, a num ber o f colonies w ere picked o ff the plate using a sterile loop and grow n in 3 ml LB (see 2.2.4.1) plus an tib io tic overnight. M iniprep plasm id DN A was p rep ared from 1.5 m l o f this cu ltu re and screened for the p resen ce o f the c o rre c t rec o m b in a n t D N A by d ig estio n w ith the ap p ro p ria te r e s t r i c t i o n e n z y m e s , a n d a n a ly s e d b y a g a r o s e g e l electro p h o resis. The rem ainder o f the culture used to m ake the m in ip re p D N A th at y ield ed a p o sitiv e clo n e, w as u sed to inoculate 500 ml o f LB plus am picillin. This culture, was grown o v ern ig h t and used to prepare a larger stock o f plasm id DNA (See 2.2.4.2) that was sufficiently pure to be used in a num ber o f applications, including transfection into em bryonic cells, DNA sequencing, in teractio n D N A -protein, etc.

A ltern ativ ely , the screening w as p erfo rm ed using one of two m ethods: G runstein and Hogness (2.2.3.1) or PCR (2.2.3.2).

2 .2 .3 C olony screening

To screen in te restin g clones, two m ain tech n iq u es w ere used: the m ethod o f by G runstein and Hogness and PCR.

2 .2 .3 .1 C olony screening by the G runstein and H ogness m e th o d

R e c o m b in a n t c o lo n ies w ere sc ree n ed a cc o rd in g to the m ethod described by G runstein and H ogness (1975).

A 0.5 cm grid was draw n w ith a pencil on circular H ybond-N ( A m e r s h a m ) m em brane the size o f a Petri dish. C olonies w ere chosen and plated both on the m em brane-circle laid on an LB- A m p icillin p late and on a rep lica L B -A m picillin p late w ith an e q u iv a le n t g rid draw n on the back. B oth p lates w ere then incubated overnight at 37°C. The replica plate was stored at 4°C.

T he m em branes w ith the colonies grow n on it w ere peeled from the agar p late and p rep ared for h y b rid isatio n by flo atin g them , colony side up, in denaturing solution (0.5 M NaOH, 1.5 M N aC l) fo r 5 m inutes and then in n eu tralisin g solution (0.5 M T ris-H C l pH 8.0, 1.5 N aC l) tw ice for 5 m inutes. The b acterial d e b ris w ere th en rem o v e d by w ip in g th e su rfa c e o f the m em brane several tim es w ith tissues soaked in 2X SSC, 0.1% (w /v) SDS. Finally the m em brane was rinsed in 2X SSC and air dried after crosslinking the DNA in a U.V. Stratalinker S tr a ta g e n e

T he m em b ran es w ere sto red at ro o m tem p era tu re u n til th ey w ere h y b rid ised .

2 .2 .3 .2 Colony screening by PCR

PC R re a c tio n s w ere p e rfo rm ed in 9 6 -w e ll m ic ro p la te s (H y b a id ), d ia g n o sin g the te m p la te D N A b e in g c a rrie d by in d iv id u a l colonies. C olonies w ere chosen and p lated on to a m u lti-w ell p late containing L B -A m p icillin and on to a re p lic a p la te w ith an e q u iv a le n t o rie n ta tio n . The re p lic a w ith LB - A m picillin was stored at 4°C. A m plification o f the sam ples in the o th e r p la te w as c a rrie d o u t in a H y b aid th e rm a l c y c lin g in stru m en t available in the laboratory.

25 cycles w ere typically perform ed as follow s: 9 5 °C fo r 1 m inute; 60°C for 1 minute; 72°C for 1 m inute, follow ed by a final extension at 72°C for 5 m inutes. A liquots o f each reaction were analysed on an agarose gel.

T h e a n n ealin g tem p eratu re fo r each PC R re a c tio n w as d e te rm in e d on th e b a s is o f th e le n g th an d n u c le o tid e c o m p o s i t i o n o f th e p r i m e r s u s i n g th e f o r m u l a T m = 4 (G + C )+ 2 (A + T )°. The PC R p o sitiv e co lo n ies w ere grow n o v ernight as described in section 2.2.4.1.

2 .2 .4 Preparation o f plasm id DNA

2 .2 .4 .1 Sm all scale plasm id preparation

3 m l LB p lu s c o n ta in in g 50)1 g /m l a m p ic illin w e re in o c u la te d w ith a single b acterial colony and the cells w ere grow n for 16 hours at 37°C with shaking. 1.5 ml o f culture was transferred to a 1.5 ml centrifuge tube and cells w ere spun for 5 m inutes at low speed, w hilst the rem ainder of the cu ltu re was sto re d at 4°C . T he c u ltu re m ed iu m w as rem o v e d an d the resu ltin g b acterial p ellet was resuspended in 100 |Lll solution I (50 mM glucose, 25 mM Tris-H Cl pH 8.0, 10 mM EDTA), follow ed by 200 |l l of freshly prepared solution II (0.2 N N aO H , 1% (w/v) SD S), and the bacterial debris w ere precipitated w ith 150 |I l of so lu tio n III (3 M p o tassiu m acetate, pH 4.3) on ice fo r 10 m in u te s .

A fter spinning in a m icrofuge for 5 m inutes at top speed, th e s u p e rn a ta n t w as rem o v ed in to a n o th e r m ic ro fu g e tu b e, tak in g care n o t to tran sfe r any o f the deb ris. P ro tein s w ere r e m o v e d b y e x tr a c t i o n w ith an e q u a l v o lu m e o f p h en o l/ch lo ro fo rm and DN A was precip itated by the addition of 2.5 volum es o f BtOH at room tem perature for 10 m inutes. The su p ern atan t was discarded and the D N A p ellet w ashed in 1 ml 70% EtO H, briefly air dried and resuspended in 50 \Xl of TE (pH 8.0). RN A was rem oved by the addition o f D N ase-free pancreatic

RN ase (20 |Xg/ml). 2-5 )Lll o f the DNA solution w ere digested with an appropriate restrictio n enzym e(s) in a total volum e o f 10-20

j ll for 2 hours and analysed on an agarose gel. F inally, glycerol stocks o f the clones of in terest w ere m ade w ith the aliq u o t of the culture that had been kept at 4°C.

G lycerol stocks were m ade by adding 0.1m l o f glycerol to 1ml of culture.

2 .2 .4 .2 L arge scale plasm id p rep aratio n

500 m l LB containing 50 |Ig /m l am p icillin w ere in o cu lated w ith 1 /1 0 0 0 volum e o f sa tu rated b a c te ria l c u ltu re h a rb o rin g the relev an t plasm id, and the cells were grow n fo r 16 hours at 37°C w ith shaking. C ells w ere h arv ested by c e n trifu g a tio n at 3500 rpm for 15 m inutes at 4°C in a Beckm an J-6B. C ell ly sates w ere prepared by alkaline lysis (Sam brook et al, 1989).

The bacterial pellet was resuspended in 10 m l o f ice cold so lu tio n I (50 mM glucose; 25 mM T ris-H C l pH 8.0; 10 mM E D T A ), then g en tly m ixed w ith 20 m l o f fre s h ly p rep a red solution II (0.2 M NaOH; 0.1% (v/v) SDS), and incubated at room tem perature for 10 m inutes to allow full bacterial lysis. 20 m l of ice-co ld solution III (3 M potassium acetate, pH 4.3) w ere then added, thoroughly m ixed and incubated on ice for 10 m inutes to p re c ip ita te the b acterial debris.

T he p recip itate was rem oved by centrifuging at 4000 rpm fo r 15 m inutes at 4°C in a Sorval 21 centrifuge, and by filtering the su p ern atan t through four layers of cheesecloth.

The nucleic acids obtained at this p o in t w ere p recip itated w ith 0 . 6 v o lu m e s o f p r o p a n -2-o l, in c u b a te d a t ro o m tem p eratu re for 10 m inutes and then cen trifu g ed at 5000 rpm fo r 15 m in u tes at room tem p eratu re. T he p e lle t w as w ash ed w ith 70% (v /v ) E tO H , air d ried at room te m p e ra tu re and dissolved in 3 ml o f TE (pH 8.0).

The nucleic acid solution was transferred to a 50 ml C orex tu b e and high m olecular w eight RNA precipitated by adding the sam e volum e of ice-cold 5 M LiCl and centrifuging at 10000 rpm fo r 10 m inutes at 4°C in a Sorval SS34 rotor. The supernatant w as tran sferred to a fresh corex tube, and the p lasm id D N A p rec ip ita te d at room tem perature for 10 m inutes w ith an equal volum e o f pro p an -2 -o l. A fter cen trifu g in g again at 10000 rpm for 10 m inutes at room tem perature the p ellet was w ashed w ith 70% (v/v) EtOH, briefly air dried, and resuspended in 500 |LLl of TE (pH 8.0).

T h e re m a in in g c o n ta m in a tin g R N A w as re m o v e d by