This experiment involves two parts: DNA analysis to detect for the Rubisco gene and
comparison of the Rubisco protein expression within the seleced plant tissues. The plant
tissues used were from the petal, stem, leaf, and root of Impatiens.
Part 1. DNA Analysis
DNA Extraction and Purification
The first step in analyzing the DNA of the tissues was extraction of the DNA
from the 4 distinct areas of the plant. The plant cell walls were broken down by grinding
the tissues and placed in a 50oC water bath with CTAB reagent to lyse the cells. The
DNA was then extracted by adding chloroform/octanol to separate the proteins and
polysaccharides from the nucleic acids. The DNA was further purified by condensing the
DNA through the addition of isopropanol and combining the condensed DNA to ethanol.
The ethanol was removed and the concentration of the DNA was determined by
measuring the absorbance of the sample at A260 on a spectrophotometer.
Real Time PCR
A real time PCR was set up to quantify the samples of DNA. Primers rbcl2f and
RBCL-fontana were used to give a 500 bp fragment. Samples with concentrations of
25ug, 50ug, and 100ug were used for each of the 4 tissue types. 20 ul reactions were set
up for each of the samples. The concentrations of the samples needed for the reaction
were calculated and then added to the appropriate amount of water to total 8.4 ul.
Original
Concentration
(ug/ul)
Dilution
Instruction
Dilution
Concentration
(ng/ul)
Amount Needed
for Reaction (ul)
Amount of
Water Needed
(ul)
1.37
2ul in 200ul
13.7
1.82
6.58
1.37
2ul in 200ul
13.7
3.64
4.76
1.37
2ul in 200ul
13.7
7.3
1.1
Stem 25ng
1.36
2ul in 200ul
13.6
1.34
6.56
Stem 50ng
1.36
2ul in 200ul
13.6
3.68
4.72
Stem 100ng
1.36
2ul in 200ul
13.6
7.35
1.05
Leaf 25ng
0.92
2ul in 200ul
9.2
2.72
5.68
Leaf 50ng
0.92
2ul in 200ul
9.2
5.43
2.97
Leaf 100ng
0.92
92
1.1
7.3
Root 25ng
3.58
20ul in 200ul
1.5ul in
1500ul
3.58
6.98
1.42
Root 50ng
3.58
2ul in 200ul
35.8
1.4
7
Sample
Flower
25ng
Flower
50ng
Flower
100ng
Root 100ng
3.58
2ul in 200ul
35.8
2.75
5.61
A master mix was made and 11.5 ul of this mix was added to each sample tube. This
included a 1 x reaction buffer (including Taq DNA polymerase, MgCl2, dNTPs, and
buffer) and forward and reverse primers.
Agarose Gel Electrophoresis of DNA
The DNA fragments obtained by PCR were separated by agarose gel
electrophoresis. A 1½ % agarose gel was used to run a 1 kb plus ladder for a molecular
weight, a blank control, and samples of 25 ul, 50ul, and 100 ul for both the petal and leaf.
These six samples were prepared for loading into the gel by adding 1 uL of tracking dye
(bromphenol blue in a 50% glycerol solution) to every 5 uL of each restriction digest.
The gel was run ¾ of the way and stained with ethidium bromide.
Part 2. Analysis of Protein Expression
Protein Isolation and Analysis
Protein was extracted from each distinct area of the plant by grinding 2g of the
tissue with 1.5ml extraction buffer ( PMSF added to Tris/EDTA Buffer pH=7.4 with
0.1% SDS). A Bradford assay was done to determine the amount of protein isolated.
Standards were used to generate a standard curve so that sample protein concentrations
could be predicted by knowing their absorbances. Knowing the sample concentrations of
the proteins was needed to continue the steps in western blot protocol.
Western Blot Procedure
A native PAGE (polyacrylamide gel electrophoresis) was used to separate the
proteins isolated from the tissue layers. 10% Ammonium Persulfate and TEMED were
added to Running Gel Buffer with Acrylamide to make the running gel. Overlay buffer
was used to allow the acrylamide to polymerize. 10% ammonium sulfate and TEMED
were added to Stacking Gel buffer to create the stacking gel. 3 x loading dye was added
to tubes, each containing protein from a different tissue sample. The samples and a
molecular weight were loaded into the gel and run at 200 volts for 45 minutes in a 1x
running buffer. Two of these gels were run. The bad gel was stained with Comassie blue
while the good gel was used for the Western Blot. The good gel was soaked in 1 x
Western Transfer Buffer. A Gel Sandwich was prepared and electrophoresed in order to
transfer the gel to a membrane. The membrane was placed in a blocking solution of
TTBS and 5% nonfat dry milk.
Antibody Detection and Western Blot Analysis
Next, two antibodies were used to detect for Rubisco. The membrane was washed
with TTBS in order to remove the blocking solution. The membrane was placed in a
blocking solution containing a primary antibody (an anti-Rubisco antibody), washed, then
incubated in a secondary antibody (anti-chicken alkaline phosphatase) and washed again.
The membrane was immersed in a color development solution.