Procedures for SDS DNA extraction method
1. 0.15 to 0.2g of fresh
plant material was collected in a moter
2. They were cut with a
scissor and grinded into fine pieces by the aid of liquid nitrogen which was carried out by me accurately well
so as prevent the negative effect of liquid nitrogen
3. Homogeneted mixture of the
plant material was then transferred into a 1.5µl eppendorf tube
4. 800µl of SDS buffer was
measured and then transferred into the grinded plant material in the eppendorf
tube. This is to aid lysing of the lipid membrane
5. Extra 200µl of the SDS
buffer was added and mix well with hand
6. The eppendorf tube
containing the mixture was centrifuge for 5min @12000rpm at a temperature
of 4ºC
7. After which the
supernatant was transferred into a new eppendorf tube
8. 200µl of 5M potassium
acetate was then added to the supernatant
and this is to separate the DNA
and RNA from other nucleic materials such as protein
9. Equal volume of
phenol-chloroform-Isoamyloalcohol in the ratio of 25:24:1 was now added into
the supernatant and then centrifuge also for 12000rpm at a temperature of 4ºC
for 10min
10.
The supernatant was then transferred into a new eppendorf
tube without disturbing the interface
11.
800µl of chilled absolute alcohol or isopropanol was then
added into the supernatant and it was kept on ice for 20min. This is to enable
the DNA molecule to precipitate since DNA molecule are insoluble in ethanol
12.
Then it was centrifuge for 10min @ 12000rpm for 4ºC
13.
The supernatant was now discarded as the DNA and RNA had
aggregated and formed a residue which
now settled at the bottom of the eppendorf
tube
14.
200µl of 70% ethanol was used to wash the DNA pellets. This
is to remove salt like potassium
15.
The pellets were left for 30-40min and allow to air dry
until the alcohol had completely evaporated
16.
The pellets were now
treated with RNase (an enzymes) which help in digesting the RNA leaving only
the DNA
17.
The pellets were now suspended in 200µl TE (Tris
ethylenediaminetetraacetic acid). The presents of the Tris is to keep the pH of
the DNA constant and the EDTA serve as a chelating agent therefore chelate ions
and some enzymes from degrading the DNA.
18.
The suspended pellet was then stored at -20ºC
