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31 Estimation of Trypsin Inhibitor in Forages (Roy and Rao 1971)
Estimation of Cyanogenic Glycosides
Estimation of Cyanogenic Glycosides (AOAC 1995b)
1. Filter paper strips
2. Picric acid solution (1%)
3. 10% Sodium carbonate (Na2CO3)
• Dip filter paper strips in 1% picric acid solution and after drying, further dip into
10% Na2CO3 solution and dry again. Store the strips in stoppered bottle.
• Place the sample of plant material in test tube. Insert a piece of moistened
sodium picrate paper in tube while taking care that it does not come in contact
with the sample. Add few drops of chloroform and stopper tube hermetically.
The sodium picrate paper gradually turns orange and then brick red if plant tissue
contains cyanogenic glycosides. The rapidity of change in colour depends upon
amount of free HCN present.
• This test works well with fresh plant materials, but relatively dry substances
particularly seeds of various plants should be ground and moistened with H2O
and allowed to hydrolyse in stoppered test tube containing sodium picrate paper.
Titrimetric Method for Quantitative Test
Acid titration method
Apparatus and Glassware
Micro-Kjeldahl distillation apparatus
Silver nitrate (0.01 N)
Nitric acid concentrate
0.02 N Potassium cyanide (KHCN)
Ferric alum indicator
Place about 10–20 g finely ground sample (sieve no. 20) in 800-mL Kjeldahl
flask. Add 100 mL H2O and macerate at room temperature for 2 h. Further add
Nutritional Evaluation of Forages
100 mL H2O and steam distil for collecting distillate in 20 mL 0.01 N AgNO3
acidified with 1 mL HNO3. Adjust the distillation apparatus so that tip condenser
dips below surface or liquid in receiver. When 150 mL distillate is collected then
it is passed through Gooch crucible. Wash the receiver and Gooch with little H2O
and titrate excess AgNO3 in combined filtrate, and washings with 0.02 N KH CN,
using ferric alum indicator.
1 mL 0.02 N AgNO3 ¼ 0.54 mg HCN
Alkaline titration method
Distillation apparatus (micro)
0.5 g sodium hydroxide (in 20 mL H2O)
Ammonium hydroxide (6 N)
Potassium iodide (5%)
Silver nitrate (0.02 N)
• Place about 10–20 g of finely ground (sieve no. 20) sample in 800-mL Kjeldahl
flask and add 200 mL H2O and allow to stand for 2–4 h (analysis is done with
apparatus completely connected for distillation). Now steam distil and collect
150–160 mL distillate in NaOH solution (0.5 g in 20 mL H2O) and dilute to a
• To 100 mL distillate (preferably dilute 250 mL and titrate 100 mL aliquot) add
8 mL 6 N NH4OH and 2 mL 5% potassium iodide solution and titrate with 0.02 N
AgNO3 using micro burette. The end point is faint but permanent turbidity, which
can be easily recognized, especially against black background.
1 mL 0.02 N AgNO3 ¼ 1.08 mg HCN (1 Ag equivalent to 2 CN)
Qualitative Estimation of Ricin
Qualitative Estimation of Ricin (Olsnes et al. 1974)
The ricin present in saline extract of castor (Ricinus communis) bean meal (CBM)
exerts haemagglutinating activity and can be utilized for detecting the presence of
CBM in feeds.
1. Normal saline (0.9%, NaCl)
3. Solution containing 0.14 M sodium chloride, 20 mM sodium phosphate per
100 mL (pH 7.1)
4. Bovine serum albumin
Preparation of crude extract
Stir 50 g of sample mechanically in 150 mL normal saline (0.9%, NaCl) for 1 h.
Filter the contents through muslin cloth and centrifuge the filtrate at 2,000 rpm for
5 min. An aliquot of clear supernatant is further tested for haemagglutinating
activity of ricin by comparative qualitative test using plate agglutination technique.
Plate agglutination test
Collect 10 mL of blood from experimental animal in equal volume of normal saline
having EDTA (1 mg/mL) blood and centrifuge at 2,500 rpm for 20 min to sediment
red blood cells (RBC). Wash RBCs thrice with normal saline through centrifugation
and dilute further with normal saline to a final suspension of 1%. Carry out
microhaemagglutination test in Lambro plate.
To each well add 100 mL of a solution containing 0.14 M sodium chloride,
20 mM sodium phosphate (pH 7.1) and 100 mg BSA per mL. Add 100 mL of the
crude extract to the first well and make serial dilutions by transferring after
thorough mixing, 100 mL to the next well and so on. Subsequently, add 25 mL of
normal saline washed 1% erythrocytes to each well and mix gently. Incubate the
plate at 37 C and record the reading after 10 min. In case of agglutination, the
erythrocytes will be sticky and cover the bottom of the well as a thin film or matrix,
whereas nonagglutinated RBCs settle at bottom in the centre of the well. Express
haemagglutinating activity as HA unit (the reciprocal of the end point dilution).
Techniques in Molecular Biology
Polymerase Chain Reaction (PCR)
(Mullis et al. 1986; Palumbi 1996)
PCR has been the most important invention of the past decade which has
revolutionized the field of molecular biology. Beginning with a single molecular
of DNA, the PCR can generate billions of copies of DNA in few hours, i.e. Nano
gram(ng) of DNA can be amplified to get mg of DNA by using this technique. PCR
technique is based on in vitro enzymatic amplification of a specific target DNA
sequence in a cyclic process using two oligonucleotides. These oligos used as
primers have different sequences and are complementary to the sequences on the
opposite strands of the template DNA and flank the segment of target DNA that is to
be amplified. Thus, given a particular target DNA, large amounts of that product
and only that product are produced in sufficient quantities for subsequent experimental analysis.
Solutions and Reagents
Upstream and downstream oligonucleotide primers
Taq DNA polymerase (5 U/ml) and 10Â PCR buffer
MgCl2, 25 mM
dNTP mix (10 mM of each dNTP)
1. Combine the first five reaction components in the order listed below in a thinwalled (0.2 or 0.5 ml) reaction tube and vortex for ~10 s and briefly centrifuge in a
microcentrifuge. Initiate the reaction by adding the template and primers
R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,
DOI 10.1007/978-1-4419-9785-2_15, # Springer Science+Business Media, LLC 2011
Techniques in Molecular Biology
Table 15.1 Reaction mixture
1. Nuclease-free water (to a final volume of 50 ml)
2. 10Â PCR buffer
3. dNTP mix (10 mM of each dNTP)
4. Taq DNA polymerase (5 U/ml)
5. 25 mM MgCl2
6. Downstream primer
50 p mol*
7. Upstream primer
50 p mol*
8. Template DNA
* The general formula for calculating nanograms of primer equivalent to 50 pmol is: 50 pmol
¼ 16.3 ng Â no of bases in promer
**Keep the final DNA concentration of reaction <10ng/ml
2. Place the tubes in a controlled temperature heat block and protocol with thermal
cycling profile chosen for the reaction.
3. Analyse the PCR reaction products by agarose gel electrophoresis (1.2–1.5%) by
loading a part of the aliquot.
4. Store the reaction products at À20 C until needed.
PCR Hygiene (Precautions)
Because PCR products are so concentrated and easily volatilized (by opening a
microfuge tube or pipetting, for instance), cross-contamination of samples is
potentially a serious problem. Certain simple precautions can be taken to avoid
contamination or at least minimize it if it occurs.
• Aliquoting solutions makes it possible to contain and help resolve contamination
problems that do arise. Each person working in the lab should have his or her
own set of solutions. PCR reagents prepared in large amounts should be
distributed in 1.5 ml microfuge tubes and stored at À20 C.
• Water used for PCR reagents, DNA, and primers should be double-distilled,
sterilized, and then distributed in 1.5 ml microfuge tubes and stored at À20 C.
• When primers are made, the stock solutions usually are highly concentrated.
From this highly concentrated stock solution, it is desirable to make a 100 mM
stock solution which can then be used in making 10 mM solutions for individual
use. The different stock solutions are stored separately. In this way, massive,
laboratory-wide contamination problems can be avoided and any contamination
problems that do arise can be contained.
• Different sets of pipettes should be designated for different procedures. One set
of pipettes should be designated for preparing PCR reactions. These pipettes
should never come in contact with any amplified DNA. Another set of pipettes
can be designated for post-PCR use. One pipette should be designated to be used
only in loading samples in agarose gels. Another set of pipettes should be
designated for use with radiation only.
Polymerase Chain Reaction (PCR)
Common Problems with PCR
Problem: No PCR product, not even in positive controls.
– Repeat the experiment.
– Check buffer, dNTPs, and primer recipes and concentrations. Remake any
– Try a different set of primer or a different positive control.
– Try a new batch of enzyme (this is seldom the problem unless the enzyme is
– Was oil added to the reactions?
– Check the thermal cycler by watching it go through 2–3 cycles.
Problem: Positive control works, but there is no product
– Run 5 ml of the stock DNA solution on 1% agarose gel. If there is a large amount
of high-molecular-weight DNA, try diluting the starting template DNA (try
dilutions of 1:10 or 1:100). If there is no high-molecular-weight DNA, increase
the amount of starting material or switch to better samples of genomic DNA.
– Try lowering the annealing temperature in the PCR cycle.
– Try a step-up cycle.
– Try using more cycles on the PCR machine (increase from 40 cycles to 50 cycles).
This is effective only when the product is present but in small quantity.
– It is possible that something in the DNA temperature is interfering with the PCR
reaction. This can be determined by setting up a single reaction with two
templates (the added template should be known to work well with the primers
being used). If the problem template prevents the added template form
amplifying, then there is something in the problem template solution that is
inhibiting the reaction. To solve this problem, try diluting the problem template,
or try one of the rescue procedures outlined above.
– Switch primers and try again.
Problem: Bright bands in well of agarose gel following electrophoresis.
Such bands usually result from overamplification of the PCR product or from
insufficient dilution of the product prior to electrophoresis. This is also a common
result of amplifications from too much genomic DNA. Try diluting the template
Problem: Smearing of double-stranded PCR products or multiple bands following
– Try less template. The most common cause seems to be too much template.
Techniques in Molecular Biology
– Try annealing temperature 2–3 C higher. A lot of smearing, or multiple bands,
may indicate that the primer is annealing to other parts of the template DNA.
– Try varying MgCl2 concentration results in the best bands.
– Try fewer cycles. This is often recommended, but is probably not the best
solution. While there may be less evidence of non-specific amplification,
subsequent amplification from this PCR reaction will amplify even minute
quantities of non-target DNA to visible levels (unless gel slices are used).
A better solution is to optimize conditions to reduce mis-priming (e.g. temperature and salt concentration in buffer).
– Try gel purifying the double strands (only take the brightest part of the band) and
then reamplify (with stringent conditions) the purified double-stranded product.
Problem: Bands in the negative controls.
– Often, in spite of all precautions, contamination problems occur. Once contamination becomes a visible problem, the contamination is more than one solution,
so altering one solution may not be informative. Fresh preparation of all stock
solutions is desirable.
– Wash the pipettes well, expose the tips to 10 min of UV light.
– Treat the solutions, including the primers, with UV light. Place the solutions in
plastic tubes on a UV light source and illuminate them for 10 min (less if the UV
source is a short wavelength source). This tends to break up contaminating DNA,
making it less attractive as a PCR template.
Isolation of Plant DNA (Murray and Thompson 1980)
A number of methods are available for the isolation of high-molecular-weight DNA
from plants. Generally, all methods involve removal of cell wall and nuclear
membrane from around the DNA and the separation of DNA from other cell
components such as cell wall debris, proteins, lipids, or RNA without affecting
the integrity of DNA.
One of the most widely followed extraction procedures involves the use of a
nonionic detergent cetyltrimethylammonium bromide (CTAB), which complexes
with carbohydrates and can be phenol-extracted. It is a relatively simple procedure
and is useful for the preparation of small samples of DNA needed for various
Reagents and Materials
1. 2Â CTAB
CTAB – 10 g
5 M NaCl – 140 ml
2 M Tris-Cl, pH 8.0 – 25 ml
0.5 M EDTA – 20 ml
Isolation of Plant DNA
2. Chloroform and isoamyl alcohol solution in the ratio of 24:1
3. 3 M Sodium acetate (pH 5.2):
Sodium acetate ¼ 408.1 g + Sterile H2O ¼ 800 ml
Adjust pH to 5.2 with glacial acetic acid and make up the volume to 1 l and
autoclave the solution before use.
4. DNase-free RNase A:
RNase A (10 mg/ml) in 10 mM Tris (pH 7.5) + 15 mM NaCl
Heat to 100 C for 15–20 min to make it DNase free and cool slowly to room
temperature (RT). Store in small aliquots at À20 C.
5. Proteinase K: 20 mg/ml proteinase K (store at À20 C)
6. 25% SDS
SDS – 25 g
Sterile H2O – 100 ml (warm to dissolve)
7. TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0
Tris – 1.211 g
EDTA – 0.372 g
Sterile H2O – ~800 ml
Adjust pH to 8.0 and make up the volume to 1 l with sterile H2O
1. Material for DNA extraction
Procure the seed material which will be the source of DNA. Wash the seeds
thoroughly with distilled water and then with 50% ethanol for 10 min. Soak the
seeds in 0.001% mercuric chloride for 10 min and then wash them several times
with distilled water and soak them overnight. Keep the seeds for germination on
wet germination towels at the desired temperature and humidity till they grow
3–6 in. in height. Cut the seedling 1 in. above the surface to minimize the
bacterial contamination and cut them into smaller pieces. Weigh them and
store them after a dip in liquid N2 at À20 or À70 C for further use.
2. Isolation of plant DNA
1. Add b-mercaptoethanol (b-ME) to the required amount of 2Â CTAB
extraction buffer to a final concentration of 0.2%. Heat b-ME/CTAB solution
to 65 C in a waterbath for 5 min.
2. Grind 10 g of etiolated seedlings in liquid N2 to a fine powder with a pestle
and mortar. Be very careful while powdering the tissue as the mortar and
pestle can shatter due to the extreme cold.