Isolasi DNA RNA Elktroforesi1
Isolasi DNA RNA Elktroforesi1
RNA
Oleh :
Dra. Fatchiyah, M.Kes. Ph.D
2/28/2011
fatchiyah
TABM JB UB
2/28/2011
fatchiyah
TABM JB UB
2/28/2011
fatchiyah
TABM JB UB
Extraction/Precipitation Method
Step 1: Disruption of cell walls by grinding
Step 1+2: mechanical disruption and
homogenization in extraction buffer
fatchiyah
Crude lysate
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Penghilangan protein/ekstraksi
fenol
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TABM JB UB
2/28/2011
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TABM JB UB
Larutan stok
Na asetat
NaCl
5,0 M
Amonium asetat*
4,0 M
1,3 M
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*sterilisasi menggunakan filter,
jangan diotoklaf
Konsentrasi akhir
Sifat
pH
Tris
Pengaruh
Penghambat enzim
degradative
bufer
Ethylene diamine
tetraacetate (EDTA)
Menjaga pH
Mengkelat kation (Mg),
menghambat enzim yg tgt
logam (nuklease, DNase)
Na/K
garam
Menstabilkan inti
Proteinase K
enzim
Mendigest protein
Sodium dodecyl
sulfat (SDS)
Detergent
anionik
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Komponen
Sifat
Pengaruh
Cetyltrimetilamonniu
mbromide (CTAB)
Detergen
kationik
Chloroform-isoamyl
alkohol (CIA)
Ekstraksi protein
Isopropanol)
Hidroksikuinolin
Glukosa, cystein
Agen
pereduksi
-merkaptoethanol
Glutation, Na2S2O5
Dithiothreitol
Asam askorbat
2/28/2011
fatchiyah
TABM JB UB
Komponen
Sifat
Pengaruh
Polyvinilpirolidon (PVP)
Absorben
polyfenol
Poliamin
Melindungi dari
RNasemencegah oksidasi
metabolit sekunder
dengan cara mengikat
senyawa fenolik.
Spermine
Spermidin
Dietylditiocarbamic
acid (DIECA)
Inhibitor
Mengkelat Cu2+
fenoloksidas
e
Cyanide
NH
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Melindungi dari H+
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Extraction/Precipitation Method
After
After
Centrifuge
Wash
Supernatant
70%
EtOH
Centrifuge
Pellet
Add alcohol and
salt to precipitate
nucleic acids
from the aqueous
fraction
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Dissolve
pellet
(H2O, TE,
etc.)
11
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RNA
(degraded)
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Plasmid Isolation
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Sodium hydroxide
denatures both plasmid
and chromosomal DNA
into single strands.
Chromosomal DNA
separates completely into
individual strands.
The single-stranded
plasmid loops remain
linked together like
interlocked rings.
Source: DNA Science: The First Course in Recombinant DNA Technology
2/28/2011
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Isolasi RNA
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fatchiyah
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Add DNase
+ DNase (protein)
Add RNase
+ RNase (protein)
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fatchiyah
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25S
18S
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More preparation
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Electrophoresis
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DNA Migration
S
large
moderate
small
Picture of DNA
separation by gel
electrophoresis
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DNA Electroelution
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DNA Electroelution . .
Electroelution is more time consuming
than some of the other techniques,
but works well and is probably the
best technique for recovery of large (>
5 kb) fragments of DNA.
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TERIMAKASIH
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