Lab 4. Molecular Cloning of Viral DNA Fragments in a Bacterial Plasmid Vector
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1 10/22/09 Lb 4. Mu Cg f V DNA Fgs Lb 4. Mu Cg f V DNA Fgs B Psd V Rdg: Appdx VIII. Rs Ezys Bgy 6 h d. By Cpb. DNA Cg p Ths b xs gy fws h squ f ubd sps shw Cpb s Fg. 20.3, wh h fwg xps: 1. Ahugh w us b psd DNA v (puc19) s h dsbd h x, u gsu DNA w b h g f b vus d Lbd (λ) h h hu DNA. Th λ Bphg P (V) DNA s pkgd h shd "hd" 2. Yu bg h xs wh psd v DNA d Lbd gsu DNA h hv dy b sd d pufd. 3. Rh h dfyg g f s u b s, w w us s dgs yss d h sz (dy) f h d Lbd DNA fg. Fgu
2 Lb 4. Mu Cg f V DNA Fgs 10/22/09 Sv fus f hs xs fquy vkd sudsd. T z fus, y kp h fwg ps d: Th psd v s y ; s y h Lbd DNA h w sd s bj f sudy. Th psd v s fu ss fus: I. Og f p ws h psd DNA p E. hs II. Ap ss g g f zy h dgds h b p E. yg psds h xpss hs g p-ss E. s h hv qud suh psd b sd by pg d g h b III. Uqu s s/s z H d I II s ss h u y h psd ug suh ss ps h u psd fu-gh f u z g IV. Z g Ap Rss puc kb Fgu 2. Og f Rp Spfd Mp f Psd puc19 DNA. squ qud f xpss f h zy b (β)-gsds β-gsds vs h f subs X-G bu-d pdu f fg DNA s sd h psd uqu s s Z, h g w b dsupd by s v hs ws sg f b psds yg fg DNA ss Th s dus y s h DNA fgs h sv s subs f g. Th zy DNA gs uy s h fgs b DNA us. 4.2
3 10/22/09 Lb 4. Mu Cg f V DNA Fgs Ahugh h b DNA us d by g f s fgs v (usd vg ), hy us b dud vg s ( vv) f p (pf). Th ss w s f s yg dff fgs f h Lbd g. Ths w su Lbd g bth b xs s bk dw 8 pdus (I-VIII) h d squy v fv b pds. Th su w gv yu h ds shdud f h pdu hs qu. W h h ppp bxs f Tb 1. Tb 1. Shdu I. Rs Dgs #1 II. Ags G #1 III. Lg IV. Tsf V. Iu d Gwh VI. Psd DNA Is VII. Rs Dgs #2 VIII. Ags G #2 IX. Ayss f Rsus Lb Rp Du DATE 4.3
4 Lb 4. Mu Cg f V DNA Fgs 10/22/09 I. Rs Dgs (#1) f Lbd DNA d puc19 DNA Th pufd Lbd d puc19 DNAs hv b puhsd. Yu dgs h puc19 d Lbd DNAs spy wh h s s zy (Hd III) d u gs g ds h h dgs hs b sussfu (Pdu II). Wh h g s ug, yu s up h g (Pdu III). Th dgs sup s pd, s b su yu udsd hw s s up (S Tb 2. d Fgu 3.) Tb 2. Rs Dgs #1 Sup C. #1 UNCUT puc19 #2 CUT puc19 #3 UNCUT Lbd #4 CUT Lbd 2X Buff 2X 2.4 µ 5 µ 2.4 µ 5 µ puc 19 DNA 0.1 µg/µ 1.6 µ [0.16 µg] 4 µ [0.4 µg] Lbd DNA 0.2 µg /µ 1.6 µ [0.32 µg] 4 µ [0.8 µg] Hd III 10 U/µ 1 µ 1 µ TOTAL 4 µ 10 µ 4 µ 10 µ Yu y fd hs b s us f ws d us w swhd. If s, f h b yu bk s h yu f b. Th 4 ubs. Tubs #2 d #4 dgss f puc19 DNA d λ DNA, spvy. Tubs #1 d #3 k dgss wh s zy s usd. N h h us f DNA usd ubs #2 d #4 ( dgss) g h h us usd ubs #1 d #3. Ths s bus DNA f h dgss w b usd f yss by phss AND f g. DNA f h k dgss w b usd y f yss by phss. 4.4
5 10/22/09 Lb 4. Mu Cg f V DNA Fgs puc 19 DNA LbdDNA HdIII AGAROSE GEL #1 HdIII puc 19 DNA fgs LbdDNA fgs Lg Mxu Tsf Pdu 1. Add h gs xp h s zy (Hd III) yu ubs h d shw h b. (Thk bu wh s ssy/ ssy hg pp ps.) 2. Mx h gs wh yu pp p. (I y b ssy sp h ubs bfy p vyhg h b.) 3. Ask h su dsps h s zy. 4. Gy x h ubs. (I y b ssy sp h ubs bfy p vyhg h b.) 5. Iub h ubs 37 C w bh f 45 us. 6. Pp 0.7% gs g (Appdx II) wh h dgs s ug. 4.5
6 Lb 4. Cg V DNA Fgs 10/22/09 II. Ags G Ephss #1 Yu shud hv yu g dy d by h h s dgs s fshd. S Fg. 4 f dg f h fwg sps. 1. Pp dgs ubs #1 d #3 y f phss by ddg 1 µ f 6X kg dy h d xg. 2. F ubs #2 d #4, v 4 µ f h. Mx h 4 µ sp wh 1 µ f 6X kg dy, sp fug ubs. 3. Kp ubs #2 d #4, wh h g 6 µ h,. Ths DNA w b usd f h g. 4. Ld h vu (5 µ) f h f h 4 ppd sps sp ws. 5. As d 18 µ f Lbd BsE II sdd DNA h ws pps sds f yu sps. Yu shud b ug DNA 6/8 s yu g. 6. Ru h g ud h s ds (100 vs ppx.) s Lb S d phgph h g. 8. Ayz h g phgph fuy. S Appdx IX. f dd gud yzg h g d pg h s dgss. Us h Lbd BsE II sdd DNA fgs su s-g p f DNA fg sz vs. g ds. Is h sz f h puc19 DNA fg ss wh h s p (Fg. 2)? A h szs f h Lbd-Hd III fgs ss wh h s p (Appdx VII)? If h bh f yu s dgss fd, yu shud s up h g wh DNA sps w pvd. 4.6
7 10/22/09 Lb 4. Cg V DNA Fgs Fgu 4. Pp f Sps f Ephss #1 4.7
8 Lb 4. Cg V DNA Fgs 10/22/09 III. Lg T g s, d hs s p dsp f hs phs f h pdu. I pdu I. yu ppd DNA fgs by dgs f h p Lbd g wh Hd III. Ths b xd wh h puc19 DNA yu ppd by dgs f h u psd wh h s zy. Wh h xu f DNA fgs s d, h fgs w ss dws du py bs-pg bw h sg-sd xss d by h s zy. Ths p bs usb bus h dvdu fgs hd gh gy by wk hydg bds. A pu hy wud gy dss sp fgs g. Th zy DNA Lgs s ddd f v bs. DNA gss ubquus zys. Thy ss pps DNA p, DNA p d b. Th vy f DNA gs bsy vss h f h s dus (Fg. 5). Fg. 5 Avy f DNA Lgss O - O - P O HO O 5' 3' SS Nk N h d py j w sp s fgs s ssy g 2 ks, h DNA sd. Th s, f u b psd ( wh fg DNA s) s ssy g 4 ks. 4.8
9 10/22/09 Lb 4. Cg V DNA Fgs Pdu 1. Lb w s fug ub d kp. 2. Csu Tb 3 f h ppp vus. Add w, buff d DNAs h ub. D dd gs. 3. P h ub 65 C w bh f 3 us, h u bh f 5 us. 4. Ask yu su dd h DNA gs yu ub. 5. P h ub 15 C f hus. Tb 3. Lg Sup C. V. Au S d W 13 µ 10X Lgs Buff 10X 2 µ puc19 Hd III dgs 0.04 µg/µ 2 µ 0.08 µg (f sp I) λ Hd III dgs 0.08 µg/µ 2 µ 0.16 µg (f sp I) DNA Lgs 1 U/µ 1 µ 1 U TOTAL 20 µ 4.9
10 Lb 4. Cg V DNA Fgs 10/22/09 IV. Tsf Rb psds h hv b d by h g sp uvb f fuh yss uss hy spd f h g pdus d pfd by p wh vg hs s. As h b p, s ds h psd d h DNA s s yg. Ths s h u g sp f h v pss. Th dsd g pdu s u psd wh sg λ DNA s h Hd III s Z. Hwv, y f dd pdus w b fd. Ud u ds h s pdu w pbby b (b) s fd by h g f h w ds f h s fg h h. Ths spy pv wh h DNA fgs h g x du. Mu g pdus b pv hgh DNA s. Th f v fgs λ DNA fgs s fu h yd f h dsd b us. Th dd sgs s h yd f b psds g x h w usg. Uy hugh, h yd f dsd pdus w v b 100%; s s wys ssy s s f vg s g h dsd pdus by s d sg. Obg pu uus f s yg spf b psd vvs:. Tsf.. E. s d wh CC 2 d h p, b k up DNA us f su. Th p s xd wh h DNA g pdus. I hs xs, yu w b pvdd wh p s. S Cs h qu psd f h g x sd b sfd d d sfs. Tsfs b gw g ps g p by vu f h p-ss g h psd. Cs whu psd ssv p. Sg Ag h s f sfd s LB + Ap + X-G p w shud bsv s bu s d s -bu ( wh ) s. Th bu s du hydyss f h X-G subs h du d s h su f xpss f h Z g fg h psd. A λ DNA s h Hd III s shud v Z, s s f sfd s yg b psd shud b bu (bus hy d hydyz X-G). 4.10
11 10/22/09 Lb 4. Cg V DNA Fgs S d sg d suusy by pg LB + Ap + X- G ps, s s sy fus h w ps. Rb h s vvs gwh vs. gwh h ps f p, bsd h ps vs. bs f psd. Sg vvs dff h pp f y bsd xpss f g (Z) h s vd by fg s. Th s, f s subj gv s (.. ds gw h ps f p) b sd. Ths s suzd Tb 4. Tb 4. S vs. Sg SELECTION SCREENING Rv fu f Ap-Rss Z G Psd V G Rv Ap X-G Cp f Ag Mdu Wh yu bsv Gwh vs. N-gwh f b y X-G hydyss (bu) vs hydyss ( bu) Wh yu B psd Psd s b 4.11
12 Lb 4. Cg V DNA Fgs 10/22/09 Pdu I hs pdu yu wk wh E. b. Rvw h hzd d sfy f f wkg wh b h ws udd h xs s dus d pg. I s u s ds dug yu pus. Yu w s up 2 fug ubs hs pdu (Tb 6). Tub #1 s h u sf wh p E. s xd wh h DNA f yu g (Sp III). Tub #2 s wh DNA. Ths dss h h p-ss s ps gy h p s. (W s h wud f h h b ps h DNA g xu. W d hs sv g ps d DNA.) Tb 6. Tsf Sup #1 + DNA #2 - DNA C S dh 2 O 20 µ DNA f g 20 µ Cp Cs 50 µ 50 µ TOTAL 70 µ 70 µ 1. Rv yu g x f h 15 C ub d kp. 2. Thw ub f p s. Thwg ks bu 10 us; us wh 30 us f hwg. D h s ud. 3. Add gs ubs #1 d #2 dg Tb Iub h ubs f 30 us. DNA bds h suf. 5. H shk h sf f 90 sds 42 C. Us h 42 C WATER BATH. DNA s h s. Cs fg hs p hd gy. B fu js ubs. 4.12
13 10/22/09 Lb 4. Cg V DNA Fgs 6. Add 0.93 LB du h ub. N h h s NO ANTIBIOTIC hs du. 7. Iub h ubs 37 C f ppxy 45 us. Ahugh y f h psd DNA h s s h gyp dy, ub pd s qud w f xpss f h b ss g h psd h phyp. Th ub s sh w sgf dvs. Lg s dvd s udsb bus ds vs f sf ffy. Th vu f hs uu, 1, w b usd s h bss u sf ffy (s Sp V). Pp fwsh suzg sps 8 d 9 bf ss d hv hkd bf pdg. 8. Du uu #1 f pg. Pp 10-1, 10-2, d 10-4 dus f h sfd s ub #1 usg s s (0.9% w/v NC) s du. 9. P s P 50 µ sps f ub #1 LB + Ap + X-g g ps dus f 10 0, 10-1 d P 50 µ sp f ub #2 LB + Ap + X-g p du f P 50 µ sp f ub #1 LB + X-g p (whu Ap) du f Th pg p s suzd Tb 7. Tb 7. Pg f Tsfd Cs Sp #1 +DNA Sp #2 -DNA LB X-g 10-4 LB Ap X-g 10 0 ; 10-1 ; Us h-sd, fd b d spd hs suspss. Af wg qud sk (f hs s vus, ud 10 us ss), v ps d ub hus 37 C. 4.13
14 Lb 4. Cg V DNA Fgs 10/22/09 Chk: yu ubg 4 LB Ap X-g ps d 1 LB X-g p, gh? Yu su w gv yu spf ds gdg vg h g ps f h ub. 4.14
15 10/22/09 Lb 4. Cg V DNA Fgs 4.15 S g y f S d S g f T s f d C s E. D H? A S p L - A p S N G w h N G w h N G w h G w h G wh X - G S g B u W h p U C 1 9 L b d - d g s - - g - s f x u f g p d u s s f g s D N A u q u d b y - p v p s d b p s d L b d f g u L b d f g N C O MP ET EN T
16 Lb 4. Cg V DNA Fgs 10/22/09 V. Iu d Gwh f Rb d N-Rb Tsfs 1. Ex h g ps f Phs IV. Rd yu sus d, bf pdg, b su yu udsd h bss f h sus yu bsvd. Wh s h vd h yu hv sfd s? Whh p/s shud hv s f s g b psds h b sd? 2. Pp 2 s ubs wh 3 LB + Ap qud du h ub. B su yu us dqu s sf hqu. Th p h du s psv s f s h h psd. 3. Tsf sg s h ubs f LB + Ap qud du. Us s wd sks. Pk wh y d bu y. Iu y p ub. 4. Iub 37 C. Pu h ubs shkg w bh fd shk/ub 37 C f hus. Fwg ub, h uus shud b sd 4 C, f ssy, u h phs VI. Yu su w gv spf sus gdg v f h uus f h shk. 5. Cu h s h g ps. B su h yu d h ub f bu d wh s spy. If h h 500 s p yu shud s. Th ps b kp h fg f yss. 4.16
17 10/22/09 Lb 4. Cg V DNA Fgs Ayss f Tsf Th bu d wh s h yu sd h LB + Ap ps us psd DNA. (Why?) Yu w yz h sf s hw w wkd. Th ffy f sf dpds h quy f yu p s d h u f h DNA. If s f h psds yu hv sfd h b d fg DNA, h h DNA hs b d, g h sg p w sds hughu sg b d b pfd s uh s yu k spy by gwg h b uu. Th X-g sg hqu w sg h ps f fg DNA h v, bu ypy ws uy s h s bsuy vd s ps d d s sz. Ths s sg by ug u f h psd. Yu w d hs h subsqu sps f hs by xs. Th sv wys su yu sus. W w u:. h fquy f s g h ppu f p s h b sfs. h ub f sfd s p µg f DNA (Ths s h sdd su f sf ffy.). h % f sfs h y b psds HINTS: I w b v bs h us h 1 uus sp IV.7. Th us w b s hs yu pfd h E. pg xs (Lb 3). Sv py us ssy: h TOTAL ub #1. h f sfs ub #1. h DNA ub #1. My vbs w ff h ffy f sf, udg h f f h DNA. F supd psds, ffs bw 10 6 d sfs p µg f DNA gy xpd. Lg pdus suh s yus xd (.. supd) d gv uh w sf ffs. 4.17
18 Lb 4. Cg V DNA Fgs 10/22/09 VI. Is f Psd DNA f E. usg h Pg Wzd TM DNA Puf Sys I d f h yu hv d b psd g Lbd DNA s, d dfy whh Lbd DNA fg/s sd, s ssy s h b psd DNA f sfd s d subj s yss. As ps, h -b psd w s b yzd. Th qud uus yu ud Sp IV w pvd h psd DNA s f yss. Th s f psd DNA f s s pqus f fuh pu d sudy, udg squ d d s yss f h d DNA fg. S-s (< 10 f b uu) ss f psd DNA, kw s pps, yd suff DNA f s yss. Spg DNA f h p, RNA, pd, bhyd d h sd by yss, wh zg DNA dgd, s v bh sk. Spg psd DNA f h buk f E. hs DNA s v dffu. Ths psd DNA s hqu ks us f h f h h psd DNA s f h b yps, wh h b hs s hd h b. I ys, b fgs wh hd hs DNA ppd by s h Th psd DNA s su. Th Wzd Mpp DNA Puf Sys ( pdu f h Pg Cp.), pvds sp d b hd f pdy sg psd DNA. Ths sys wks s ffy wh psds <20 kb. Th pss b pd s s 15 us. Psd DNA ppd by hs sys s f suff puy h b usd dy f s dgs sf whu fuh puf. Pg Wzd pps s bu $ 0.75 p DNA s. Th j s s h ppy DNA bdg s. Th Wzd ks f hs by xs hv b dd by h Pg Cp. Th yd f psd DNA w vy dpdg ub f fs, udg h vu f b uu, psd py ub (# f psd us/), yp f uu du d h b s. puc19, h psd usd hs xs, hs py ub f 500 psds p d yds f ppxy 5 µg DNA / uu yp. N h g ss (>8 kb) b dvvs f puc19 y w h py ub subsy. Ths w usuy b fd w DNA yd. Th x py ub d yd f pu b psd b pdd wh y. 4.18
19 10/22/09 Lb 4. Cg V DNA Fgs G Suy f pdu: Lyss f s by xpsu hgh ph d dg. RNs zy s ps du RNA. ph uz uss pp f w dbs W dbs wh hd hs DNA s pd d dsdd. Bdg f psd DNA sub s. Eu f psd DNA f s f wshg. Fuh f h Pg Wzd DNA Puf Sys s vb :
20 Lb 4. Cg V DNA Fgs 10/22/09 Pdu Pp fw dg f hs pdu bf g ss d hv hkd bf pdg. Ahugh h pdu s wdd s f yu w dg pp, yu w b dg 2; f h uu gw f wh sf d h h f h uu gw f bu sf. Thus, yu d p sps bw dup. B fu x up h w pps. N h s hqu s qud hs pdu. 1. Tsf 1.2 f vgh uu fug ub. Ag h uu fs s h s hv sd h b f h ub. 2. Sp fug f u xu spd p h s. B yu ubs h. Yu p w b s bu shud b vsb h b f h ub. 3. Whdw h sup fuy wh yu P-1000 d dsd by ppg bk h uu ub f. Rv s uh f h sup s pssb whu dsubg h p. Th uu ubs bhzd ws. 4. R-suspd h p py 200 µ f C Rsusps Su (RS) (D us y h su by sk!) Thughy dsps h ups. Yu shud vx d/ dw h su pdy d u f h pp p dsps h ups.! PUT ON GOGGLES AND GLOVES THROUGH STEP 13! 5. Add 200 µ f C Lyss Su (LS) yu susps d x by vg h fug ub 4 s. Th ubdy (udss) f h susps shud ds d yu y h su hs b vsus. Vssy s gd - s yu hv bd hgh u wgh DNA f h s. LS s h dg Sdu Ddy Suf (SDS) ph Add 200 µ f Nuz Su (NS) d x by vg h ub sv s. NS s Pssu A ph 4.8. C w dbs shud f sub pp. 4.20
21 10/22/09 Lb 4. Cg V DNA Fgs 7. Cfug h ys fug f 5 us. B yu ubs. Th p w w d b fgs, wh h buk f h hs DNA hd (uss xsv shg f h DNA hs ud hugh ugh hdg). Ths p w b ss w dfd h h p sp 3 d y b sd g h sd f h ub. If p hs fd, fug f dd 15 us. Th psd DNA s h sup. Dug h sp yu bg s up f sps 8 d F h pp pp Wzd Mu s fws: Rv h pug f 3 dspsb syg d s sd. Ah h syg b h Lu-Lk xs f h Mu. 9. Ob fug ub p pp wh 1 f suspdd DNA Puf Rs (PR) h. N: Th s sf s sub d shud b vsb s udy susps d h b f h ub. Thughy suspd h DNA Puf Rs by vxg bf vg qu. Us h P-1000 sf h s susps h b f h syg/u ssby. 10. Cfuy v (0.6 ppx.) f h DNA sp f h fug ub (.. h sup f Sp 7) d sf h b f h syg/u ssby. Ty suk up y f h guk h sd f h ub. 11. Cfuy s h syg pug h b d gy push h suy d hugh h Mu. Th f us b d h sp ws v pvdd d h py fug ub us b pd h sp dy ws pvdd. A hs p, yu psd DNA shud b bud h s bds ppd h u. Th py fug ubs us b pd h sp dy ws pvdd h hd. 4.21
22 Lb 4. Cg V DNA Fgs 10/22/ Dh h syg f h Mu AND THEN v h pug f h syg b. Rh h syg b h Mu. IMPORTANT! I s h yu dh h syg f h Mu BEFORE yu v h pug f h syg. Ohws, yu w suk h s u f h Mu d bk h syg b. Ths dsys yu pp. 13. Pp 2.0 f Cu Wsh Su (CW) h b f h Mu/syg ssby. Is h pug h syg d gy push h Cu Wsh Su hugh h Mu. Th f us b d h sp ws v pvdd. Cp h ws v d s sd f pp dsps. Yu y w v yu gggs d gvs. 14. Rv h syg d sf h Mu 1.5 fug ub wh h p vd. Cfug h Mu fug f 2 us dy h s. Th fug ubs d syg us b pd h sp dy ws k pvdd h hd. 15. Tsf h Mu w 1.5 fug ub (wh p vd bu svd). 16. Add 50 µ f Ts-EDTA Buff (TE) h Mu d fug fug f 20 sds u h DNA. 17. Rv d dsd h Mu. Pd dy h s dgs (Sp VII) s h psd DNA h fug ub 4 C -20 C. Typ DNA yd: µg/µ (ppx.) 4.22
23 10/22/09 Lb 4. Cg V DNA Fgs VII. Mu Ayss f Rb Psd by Rs Ayss Usg pps, yu hv sd yu b psd DNA, d v puc19 DNA f E. s bd by sf f p s wh DNA f h g. T yz h b, s u wh Hd III d h fgs xd by g phss. Th g s dfy h bd DNA s d puc19. Yu pfd s dgss f psd DNA w pvus b pds (Lb 8 d Sp I f hs xs). I bh ss yu fwd xp pdu s fh d hs u. H w xp yu yu w pdu, bsd h gud gv bw. I shud b hpfu g bk d vw h pdu ds f h 2 pvus dgss. 1. Dvp g sgy. Hw y dff dgs ubs w yu s up? Wh DNA sps w hy d whh s zy/s w yu us f h? D fg ud s h w vfy h dy f fgs f bh pps. 2. Th fud ssu wkg u x pdu s d hw uh DNA us h dgs. Ths s gvd by wh yu w d wh f dgs. I phs I, f xp, h dgsd DNAs w usd 2 wys (ph yss d g). H, h DNA w b usd y f g phss. Yu us us ugh DNA s h h bd h g s vsb. A p u f hub ppb h s h bd gs g shud dy hv bw 10 g d 100 g f DNA. (I s wys pssb z hs d wh fg szs wh gv vy v h 10X g.) O h h hd, dgsg xssv us f DNA s wsfu. By sdg h v szs f puc19 (2.686 kb) d h vg sz f h xpd s/s, w k ugh s f h u f DNA dd f h dgs. W d ug 0.5 µg. L wh h sz f yu DNA s hs b dd, w b vy whwh f yu bk d u hw uh DNA s ps h s bd yu g. 3. Nx, d h vu f DNA sp us h dgs bsd h xpd yd fgus f h pp gv. Ths y b ughy sd. 4. D h u f s zy/s us bsd h u f DNA h dgs, h df f u vy, d h f h zy sk. Us 5 10-fd xss f zy su p dgs. Thk hd h pssby h yu y d du h zy sk. 4.23
24 Lb 4. Cg V DNA Fgs 10/22/09 5. Nx d wh h vu f h dgs w b. Rb h h xu vu yu d h ws f u g sys s 20 µ, d h yu w dd 6X kg dy h dgs bf dg. 6. D h u d u f s buff dd. Rs buff s vb s f 2X 10X. 7. D h vu f w ssy f h dgs ub bg h h s vu. 8. Suz yu pdu bu f suh s h usd dsb h pvus dgss. 4.24
25 10/22/09 Lb 4. Cg V DNA Fgs VIII. Ags G Ephss #2 Ths g dfs h Lbd Hd III fg/s yu hv d yu b psd. Ru 0.7% w/v gs g ud h s ds s pvus gs. Ru 2 DNA sdds (pvdd) dd h sps f yu dgss (u d uu; wh d bu). Th 2 sdds : 1. λ BsE II DNA 2. λ Hd III DNA IX. Ayss f Rsus If h g shws h yu hv d h Lbd Hd III fg, yu p shud f yu sd f hw hs gh hv hppd. Th sv pssbs: Th g Lbd DNA dgs dd g p d yu d f h py dgsd fgs. Yu psd s y g pdu (.. sus f g f w dff Lbd Hd III DNA fgs wh h psd). Yu qud w dff b psds, h g sg Lbd Hd III DNA fg. Hw yu dsgush hs pssbs? Yu shud s f h ss sus. W s fgs d fquy wh hs w d ss fquy? If s, why? 4.25
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