10 p i from th e above re a c tio n w as lo ad ed onto a 1.2% agarose gel along w ith (j)X174-Hae III fra g m e n t size-m ark er a n d th e p roduct sizes analysed a fte r electrophoresis. The gel w as th e n p h o to g ra p h e d u n d e r UV lig h t, d e n a tu r e d w ith a so d iu m hydroxide a n d sodium chloride so lu tio n (see so u th e rn b lo ttin g
m ethods) a n d th e n b lo tted onto e ith e r H ybond-N or Hybond-N"*" (A m ersham ) w ith o u t n e u tra lisa tio n .
pC p rim er consists of a twelve base poly dC strin g on th e 3’ end of a N ot 1 an d a n Eco R1 site, plus a few o th er random b ases a t th e 5’ end to avoid having a restric tio n site a t th e extrem e en d of th e stra n d (see Table 2.1, pages 56-58). N ot 1, being a n 'in freq u en t c u tte r ’, w as th e enzym e o f choice for one en d of th e RACE frag m en ts, w hile th e specific p rim e rs w ould e ith e r in co rp o rate a re s tric tio n site in th e ir 5’ end, or w ould be d esig n ed to p rim e across a n a tu ra l restric tio n site in th e sequence a t th e o th er end. T h is la tte r offered an y easy option for re c o n stru c tin g com plete coding regions from overlapping clones.
If th e re w ere an y am p lified fra g m e n ts of in te r e s t, th e rem a in in g PCR reactio n w ould be subjected to double re s tric tio n enzym e d ig est w ith a n excess of enzym e for j u s t one ho u r, or to two se rial d ig ests w ith a phenol a n d chloroform e x tra ctio n a n d eth an o l p recip itatio n in betw een. The resu ltin g solution w as th e n electro p h o resed on 1% LMP (low m eltin g point) ag aro se (Gibco, BRL) an d th e h a n d of in te re s t w as excised w ith a scalpel over a U.V. lig h t source.
DNA w as ex tracted from th e agarose by adding th ree to four tim es th e volume (of gel) of TE buffer an d h eatin g it to 55®C for five m in u te s a n d th e n ex tra ctin g w ith phenol, phenol a n d chloroform, a n d th e n chloroform alone before e th a n o l p re c ip ita tio n w ith sodium acetate, often w ith th e addition of tRNA.
A ltern ativ ely , 'G eneclean' (Bio 101) w as u se d to p u rify th e DN A fro m slices of LM P a g a ro se . T h is w as done s tr ic tly according to th e m a n u fa c tu re r’s in stru ctio n s.
R e s t r ic ti o n d ig e s tio n of th e P C R p r o d u c ts l e f t p h o s p h o ry la te d 3’ e n d s, a n d so th e f ra g m e n ts w e re t h e n dephosphorylated before lig atin g th em in to vector DNA w hich h a d b een digested w ith com patible restric tio n enzym es.
D éphosphorylation o f PCR product digests.
U sing C IP (calf in te stin a l p h o sp h atase), a ty p ical rea ctio n w ould contain
20 mM T ris.Cl, pH 8.0. 1 mM MgCl2
1 mM Z11CI2
-2 0 pM of DNA term ini. 0.1 u n it of CIP.
A fter in c u b a tio n for th ir ty m in u te s a t 3 7 ^ 0 , th e re a c tio n w as stopped by h e atin g to 75®C for te n m in u tes an d th e n p recip itatin g th e DNA.
The C IP (B oehringer-M annheim ) w as u sed w ith e ith e r th e supplied buffer or w ith a custom m ade one as above.