Nama: Diah Ayu Ayu Sri Utami Utami NIM: 1670121041 SGD: 5 Laporan Praktikum Biomolekuler RT-PCR RT-PCR (Reverse Tr Transcriptase-PCR) anscriptase-PCR)
Bahan dan reagen: RNA Isolation it !arri"r DNA Su#"rs$ri#t III R% %a& DNA 'olym"ras" (N%' )u**"r M+!l2 S"r,u a+aros" 100 ,# DNA l"a("r .oa(in+ (y" RNAs" ut ri,onu$l"as" inhi,itor -
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5/ *irst stan(ar ,u**"r D%% tanol a,solut" Sy,r sa*" stain Ultra#ur" (istil"( at"r Ultra#ur" DNAs" RNAs"*r""
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)u**"r %ris )ori$ D%A 3%) 1
Bahan habis pakai:
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'!R tu," 02 . - Mi$rotu," 1 15 m. - 9hit" ti#s - "llo ti ti#s - )lu" ti#s Peralatan: - 'i#"ttor satu s"t )ios )iosa* a*"t "ty y $a,i $a,in" n"tt $las $lasss 2A '!R '!R $a,i $a,in" n"t tla lami mina narr air air *lo *lo $a,in"t M"=a '!R >ort"/ Ra ta,un+ '!R Ra ta,un+ '!R ,"r#"n(in+in
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8al$on tu," 15 m. Glo;"s Mas"r %issu"
M"sin '!R 8r""<"r -20 8r""<"r -40 Mi$ros"ntri*u+asi A##a A##arat ratus us "l" "l"tr tri* i*or or"s "sis is (an #o"r su##ly Syst"m +"l (oum"ntasi ?as la,
Spesimen klinis: - !airan s"rum (ari *as" aut ! Pembahasan Sam#"l ("n+an r"asi '!R #ositi* yan+ (ianalisis l",ih lan=ut m"n++unaan m"to(" multi#l"/-n"st"( '!R Untu r"asi ini (i+unaan #rim"r *orar( D1 (an "m#at #rim"r r";"rs" yan+ s#"si*i untu "m#at s"roti#" ;irus ("n+u"@ yaitu %S1@ %S2@ %S@ DN4 '"n"ntuan s"roti#" ;irus ("n+u" (ilauan s"suai uuran #ita DNA yan+ t"r,"ntu s"t"lah ;isualisasi #a(a "l"tro*or"sis +"l a+aros"@ yani 4B2 ,# untu DN-1@ 11C ,# untu DN-2@ 2C0 ,# untu DN-@ (an BC ,# untu DN-4 'a(a #"lasanaan s"lalu (is"rtaan #"m,an(in+ #ositi* untu s"tia# s"roti#" "etode M"n++unaan "m#at #rotool yaitu 1 'rotool #"rtama a(alah "strasi RNA ;irus dengue (ari sam#"l s"rum m"n++unaan EIAam# ® >ira Mini it hasil sarin+an ,"ru#a RNA #a(a ta,un+
miro 1@5 m. "mu(ian (isim#an (i freezer #a(a -B0 R"a+"n yan+ (i+unaan : - 560 F. )u**"r A>. - 5@6 F. !arri"r RNA - 140 µ. s"rum ("n+u" -
500 F. "thanol 3C6-100 500 µ. )u**"r A91
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500 µ. )u**"r A92
2 'rotool "(ua yaitu #rotool R%-'!R 3R";"rs" %rans$ri#tas"-'!R untu ("t"si (an s"roti#" DN> Di(a#atan $DNA lalu (isim#an (i *r""<"r #a(a -20 R"a+"n yan+ (i+unaan : - Nul"as" *r"" at"r - 0@1 M D%% - 10 mM (N%' Mi/ - D2 'rim"r 310 #mol - RNA - 5x first standart buffer - RNAs" ut - Su#"rs$ri#t III R% 'rotool "ti+a m"ru#aan #rotool untu ("t"si DN> Di(a#atan DNA t"m#lat" lalu (isim#an (i *r""<"r #a(a -40 R"a+"n yan+ (i+unaan : Nul"as"-*r"" at"r 10/ Ro$h" ,u**"r
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10 mM(N%' Mi/ 50 mM M+!l2 D1 'rim"r 310 #mol D2 'rim"r 310 #mol Ro$h" %a& DNA 'olym"ras" 5 U . 4 'rotool t"rahir@ yaitu #rotool ""m#at a(alah #rotool untu +"l a+aros" "l"tro*or"sis 1 S",anya 1@2 +r a+aros" (itim,an+ lalu (imasuan " (alam rl"nm"y"r@ "mu(ian s",anya 60 m. %) masuan (an homo+"nan@ 2 .arutan (i#anasan sam#ai ,"nin+@ "mu(ian (in+inan (irasa han+athan+at uu (an s",anya 6 . sybrsafe (imasuan@ "mu(ian homo+"nan tuan+ " (alam $"taan +"l (an tun++u sam#ai m"ma(at@ !"taan sumur (il"#as (an m"masuan +"l " (alam alat "l"tro*or"sis@ "mu(ian larutan %) 1/ #"rlahan (ituan+ " (alam alat sam#ai +"l t"r+"nan+@ s",anya 4 . loading +"l $am#uran up-down ("n+an #ro(u s"roti#"@ "mu(ian masuan " (alam sumur +"l s",anya 20 .@ 4 S",anya 1 . DNA leader 100 )' s",a+ai marker " (alam sumur ,"riutnya@ "mu(ian m"lauan running #a(a alat "l"tro*or"sis ("n+an t"+an+an B0 > Arus 200 mA (alam atu 45 m"nit
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#asil dan $esimpulan
%"ni '!R (a#at (i+unaan untu m"n+am#li*iasi DNA tar+"t s"hin++a =umlahnya m"n=a(i ,anya . Reaction mixture (alam t"ni '!R t"r(iri (ari ,","ra#a om#on"n yaitu %aha#an R%-'!R yaitu isolasi DNA@ annealling (an #olim"risasi .alu untu m" m,a$a hasilnya (ilauan #a(a gel doc
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Hasil (ari R%-'!R (ilauan running #a(a a+ar "l"tro*or"sis S"t"lah running s"lama uran+ l",ih =am@ hasilnya (a#at (i,a$a #a(a gel doc Hasil #"rtama m"m#"rlihatan #a(a marker base pairs 100 (an mass 4B (it"muan "m#at #ositi* ;irus dengue (ari ("la#an marker
(an hasil "(ua #a(a marker yan+ sama (it"muan (ua #ositi* ;irus dengue (ari ("la#an marker
