DNA Isolation andGel
electrophores
is
Dr. Vipul G.
Kelkar PhD (Plant
Biotechnology)
Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth,
Dapoli College of Agriculture
Plant Biotechnology Centre
2.
Buffers for DNAIsolation
Extraction Buffer (EB):
Sr No Chemicals Molecula
r
Weight
Required quantity
(gm) to make
100mL EB
1. Tris-HCl (200 mM) 157.60 3.15 g
2. NaCl (250 mM) 58.44 1.46 g
3. EDTA (25 mM) 372.24 0.93 g
Total Make up volume up
to100 ml using
distilled water
Check pH (8.00) and autoclaved at 1210
C and 15lbs for 20 min
Working Buffer / Lysis Buffer (10mL)
1. Glucose (0.5M) (Mol weight 180.156 g/mol) 0.9 g
2. PVP (3%) 0.3 g
3. Sodium bisulphate (0.4%) 0.04 g
4. SDS (0.5%) 0.05 g
5. Sarkosyl, (Sodium Lauroyl Sarcosinate) (0.5%) 0.05 g
6. Extraction Buffer 10 ml
3.
Other chemicals andplastic wares
1. Chloroform : Isoamyl alcohol (CIA) (24:1)
2. Isopropanol (2-Propanol)
3. Ethanol (70%)
4. Eppendorf tube (1.5mL)
5. Micro pestle
TE Buffer (TE):
Sr No Chemicals Molecula
r
Weight
Required quantity
(gm) to make
100mL TE
1. Tris-HCl (10 mM) 157.60 0.157 g or 157mg
2. EDTA (1 mM) 372.24 0.029 g or 29 mg
Total Make up volume
up to100 ml using
autoclaved
distilled water
Check pH (8.00) and autoclaved at 1210
C and 15lbs for 20 min
4.
TAE Buffer (TAE-50X):
Sr
N
o
Chemicals(50X) Molecula
r
Weight
Required quantity (gm) to
make TAE-50X
100 ml 500 ml
1. Tris or Teis base (2 M) 121.14 24.228 g 121.14 g
2. EDTA disodium
salt (50 mM)
372.24 1.8612 g 9.306 g
3. Glacial / acetic
acid (1M)
57.08 5.7 ml 28.54 ml
Total Make
up
volume
up to100
ml
usin
g autoclaved
distilled
Make up volume
up to 500 ml using
autoclaved
distilled
water
Buffer for agarose gel electrophoresis
5.
1) Collect freshleaf samples 10-100 mg from 2-3
months old plants, wipe with ethanol (70%)
and crush in 1.5ml Eppendorf tubes using
micro pestle in buffer (750μl) lysis buffer and
invert tube 3-4 times for mixing.
2) Incubate the tube at 650
C for 45 min., and
keep for 10 min. at RT.
3) Centrifuge the tubes at 10,000 rpm, 40
C for 10
min. and collect supernatant to new 1.5 ml
tube and discard rest.
4) Then add CIA 500 μl and vortex it for 15 sec.
again Centrifuge the tubes at 10,000 rpm, 40
C
for 10 min.
5) Collect supernatant to new 1.5 ml tube and
discard rest.
Protocol for DNA Isolation
6.
7) Centrifuge thetubes at 10,000rpm, 40
C for 10 min.,
discard supernatant and add ethanol (70%) to
remove unwanted chemical traces.
8) Centrifuge the tubes at 10,000rpm, 40
C for 05 min.,
discard supernatant and air dry the pellet.
9) Resuspend pellet in 50μl TE buffer or sterile nuclease
free water. Keep tube at 370
C for 10 min if needed.
10) Store at 4°C for immediate use or at -200
C to be used
latter.
8.
Agarose gel electrophoresisof the DNA was done to check its
quality, while spectrophotometer readings gave an indication
of the concentration and cleanliness.
1) Make a 0.8% agarose gel with 1x TAE and 0.5μl of
ethidium bromide (10mg/ml) per 10ml solution.
2) Load samples (3μl sample + 2μl 6x Loading Dye) in the
wells, along with uncut Lamda DNA (100ng/μl of DNA)
of known concentration, for quality.
3) Run the gel for 30 min at 80 V.
4) Expose the gel to UV light under gel documentation
system. Presence of a highly resolved high molecular
weight band indicates good quality DNA, while presence
of a smeared band indicates DNA degradation.
Protocol Agarose gel electrophoresis
