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31 Estimation of Trypsin Inhibitor in Forages (Roy and Rao 1971)

31 Estimation of Trypsin Inhibitor in Forages (Roy and Rao 1971)

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14.32



Estimation of Cyanogenic Glycosides



14.32



373



Estimation of Cyanogenic Glycosides (AOAC 1995b)



14.32.1



Qualitative Test



Reagents

1. Filter paper strips

2. Picric acid solution (1%)

3. 10% Sodium carbonate (Na2CO3)

Procedure

• 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.



14.32.2



Titrimetric Method for Quantitative Test



Acid titration method

Apparatus and Glassware

1.

2.

3.

4.

5.

6.



Micro-Kjeldahl distillation apparatus

Kjeldahl flasks

Conical flasks

Burette

Pipette

Gooch crucible



Reagents

1.

2.

3.

4.



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



374



14



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.

Calculations

1 mL 0.02 N AgNO3 ¼ 0.54 mg HCN

Alkaline titration method

Apparatus

1.

2.

3.

4.

5.



Kjeldahl flask

Distillation apparatus (micro)

Conical flask

Burette

Pipette



Reagents

1.

2.

3.

4.



0.5 g sodium hydroxide (in 20 mL H2O)

Ammonium hydroxide (6 N)

Potassium iodide (5%)

Silver nitrate (0.02 N)



Procedure

• 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

definite volume.

• 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.

Calculations

1 mL 0.02 N AgNO3 ¼ 1.08 mg HCN (1 Ag equivalent to 2 CN)



14.33



Qualitative Estimation of Ricin



14.33



375



Qualitative Estimation of Ricin (Olsnes et al. 1974)



Principle

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.

Reagents

1. Normal saline (0.9%, NaCl)

2. EDTA

3. Solution containing 0.14 M sodium chloride, 20 mM sodium phosphate per

100 mL (pH 7.1)

4. Bovine serum albumin

Procedure

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).



Chapter 15



Techniques in Molecular Biology



15.1



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

1.

2.

3.

4.

5.

6.



Template DNA

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)

Nuclease-free water



Protocol (Basic)

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

(Table 15.1).



R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,

DOI 10.1007/978-1-4419-9785-2_15, # Springer Science+Business Media, LLC 2011



377



378



15



Techniques in Molecular Biology



Table 15.1 Reaction mixture

Volume



Final concentration



1. Nuclease-free water (to a final volume of 50 ml)

 ml

2. 10Â PCR buffer

5 ml



3. dNTP mix (10 mM of each dNTP)

1 ml

0.2-mM each

4. Taq DNA polymerase (5 U/ml)

0.25 ml

0.0025 U/ml

3 ml

1.5 mM

5. 25 mM MgCl2

6. Downstream primer

50 p mol*

1 mM

7. Upstream primer

50 p mol*

1 mM

8. Template DNA

y ml**

* The general formula for calculating nanograms of primer equivalent to 50 pmol is: 50 pmol

¼ 16.3 ng  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.



15.1



Polymerase Chain Reaction (PCR)



379



Common Problems with PCR

Problem: No PCR product, not even in positive controls.

Solution

– Repeat the experiment.

– Check buffer, dNTPs, and primer recipes and concentrations. Remake any

questionable solutions.

– 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

very old).

– 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

Solution

– 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.

Solution

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

100–1,000-fold.

Problem: Smearing of double-stranded PCR products or multiple bands following

electrophoresis.

Solution

– Try less template. The most common cause seems to be too much template.



380



15



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.

Solution

– 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.



15.2



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

experimentation.

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



15.2



Isolation of Plant DNA



381



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

Procedure

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.



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