RFLP/ PCR-RFLP

PCR-RFLP

   Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another. Polymorphisms are inherited differences found among the individuals in more than 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.

Principle

Restriction endonucleases are enzymes that cut lengthy DNA into short pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at different sites. The distance between the cleavage sites of a certain restriction endonuclease differs between individuals. Hence, the length of the DNA fragments produced by a restriction endonuclease will differ across both individual organisms and species.

DNA Extraction

To begin with, DNA is extracted from blood, saliva or other samples and purified.

PCR

Isolation of sufficient DNA for RFLP analysis is time-consuming and labor intensive. However, PCR using specific primers can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time.

DNA Fragmentation/ Production of Restriction Fragments

The purified DNA is digested using restriction endonucleases. The recognition sites of these enzymes are generally 4 to 6 base pairs in length.  The shorter the sequence recognized, the greater the number of fragments generated from digestion. The shorter the sequence recognized, the greater the number of fragments generated from digestion.

Gel Electrophoresis

The samples of DNA that have been treated with restriction enzymes are placed in separate lanes on a slab of electrophoretic gel across which is placed an electric field. The fragments migrate to wards the positive electrode, the smaller fragments moving faster than the larger fragments, thus separating the DNA samples in to distinct bands.

Visualization of Bands

The gel is treated with luminescent dyes in order to make the DNA bands visible.

Applications of RFLP:

RFLPs can be used in many different settings to accomplish different objectives.

RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. (i.e. it has forensic applications). RFLPs can be used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known muations). RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans. 

DETERMINATION OF TOTAL HARDNESS

Hardness is defined as calcium and magnesium ion content. Hard water contains bicarbonate, chlorides and sulphates of calcium and magnesium. The water having low level or absence of calcium and magnesium ion refers as soft water. Water hardness is usually noticed by difficulty in lathering soap because it gets precipitated in the form of insoluble salts of calcium and magnesium. Hardness of water is a measure of the total concentration of the calcium and magnesium ions expressed as calcium carbonate. When water passes through or over deposits such as limestone, the levels of Ca2+, Mg2+, and HCO3 – ions present in the water can greatly increase and cause the water to be classified as hard water. Hardness of water is one of the important water qualities measuring parameter but it does not consider as pollution parameter. Hardness usually does not harm the human health of in major way; however soft and very hard water may create the problems in human health.

There are two types of hardness

1. Temporary hardness

       Temporary Hardness is due to the presence of bicarbonates of calcium and magnesium. It can be easily removed by boiling.

2. Permanent hardness

      Permanent Hardness is due to the presence of chlorides and sulphates of calcium and magnesium. This type of hardness cannot be removed by boiling.

Hardness is usually reported as parts per million (ppm) of calcium carbonate (by weight). A water supply with a hardness of 100 ppm contains the equivalent of 100 g of CaCO3 in 1 million g of water or 0.1 g in 1 L of water (or 1000 g of water since the density of water is about 1 g/mL).

The maximum level of hardness for potable water by WHO is 500mg/L and recommended hardness is moderately hard.

PRINCIPLE

The total hardness is determined by titration with ethylenediamine-tetraacetic acid, disodium salt dihydrate (Na2EDTA). A water sample is buffered to pH 10.1 and taken in to a conical flask. On addition (erichrome black T) EBT indicator dye in solution containing Calcium and Magnesium ions turns to wine red due to complex formation between calcium and magnesium ion with EBT at pH 10.1. EDTA, the titrant, removes the magnesium and calcium ions complexes with the indicator. When all the Mg and Ca are complexes with EDTA, the indicator will turn blue. This is the end point of the titration.

Materials required

Apparatus required

1. Burette

2. Pipettes

3. Conical flask

5. 100 ml graduated cylinders

6. Beaker

Chemicals required

1. Ammonium Buffer pH 10.1

2. EDTA (Disodium Salt of EDTA) -0.02N

4. Erichrome Black T

Procedure:

Take 50 ml of sample in a clean 250mL conical flask. Add 2mL of Ammonia buffer solution to the water sample so that the pH will be maintained between 10 and 11. Add few drops (100mg) of EBT indicator to the conical flask and the sample turns to wine red in color. Titrate the sample against the EDTA solution in the burette till the blue colour appear. This is the end point of the titration. Note down the burette reading. Repeat the titration for concordant values.

Observation 

Preparation of chemicals:

1. EDTA solution (0.02N =0.01M):

Take 3.723g Na2EDTA and Dissolve completely in 1 liter distilled water completely.

2. Ammonium buffer pH 10.1:

i. Dissolve 16.9g ammonium chloride in 143 ml of concentrated ammonium hydroxide.

ii. Take 1.179g of Na2EDTA and 0.78g of MgSO4 .7H2O in 50 ml distilled water and dissolve completely.

 Mix both solution and maintain volume to 250 ml.

3. Erichrome Black T (EBT) indicator:

Take 0.4g EBT and mix with 100g NaCl and grind finely.

Significance of Hardness:

1. Scales are formed as inner coating of the pipelines prevents corrosion.

2. Absolutely soft waters are corrosive and dissolve the metals.

3. More cases of cardio vascular diseases are reported in soft water areas.

4. Hard water is useful to growth of children due to the presence of calcium.

5. Hard waters cause excessive consumption of soap used for cleaning purpose.

Bibliography:

1. APHA Standard Methods for the Examination of Water and Wastewater - 20th Edition. Method 2340 - C.

2. Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, USEPA, Method 130.2.

Genomic DNA extraction from animal tissues

Mr. Khadga Bikram Angbuhang

Faculty: Department of Microbiology                                                           

GoldenGate Intl College

Research officer: LEAD Nepal.                          

Genomic DNA extraction from animal tissues

Principle:

The isolation of DNA usually begins with lysis of tissue or cells. Lysis is carried out in a salt solution, containing detergents (SDS) and proteases (Proteinase K). SDS solubilizes the cell membrane and lipid membranes of internal organelles and denatures proteins (enzymes) which release chromosome in lysate. Treatment with Proteinase K and Phenol: Chloroform can dissociate histones and other proteins from DNA and precipitate them. Tris-HCL is a buffer, which retain constant pH; ethylenediaminetetraacetic acid (EDTA), binds metal ions. Chloroform solubilizes lipids and a lot of proteins and separate proteins and polysaccharides from nucleic acids in the cell. Chloroform is denser than water solutions and thus after spinning, Chloroform and water will separate into two distinct phases. The lower phase will be Chloroform. This is the phase that proteins and polysaccharides find most chemically attractive. The upper aqueous phase will contain your DNA. Free DNA is recovered in aqueous solution after centrifugation and concentrated by ethanol. DNA is less soluble in solutions containing isopropanol than in solutions containing ethanol. Precipitation with isopropanol is performed at room temperature to lessen the risk that solutes like sucrose or sodium chloride will be co precipitated with the DNA. DNA does NOT dissolve in ethanol (Chilled Ethanol). Cold alcohol is used to separate DNA out of water-based solutions.

Material Required:

            1. Extraction buffer: (NaCl 10mM; Tris-HCl 10mM; EDTA 10mM, pH 8.0)

2          2. SDS - 10%.

3. 3 M Sodium acetate, pH 4.8 by acetic acid
4. Phenol: Chloroform (1:1)
5. Chloroform: isoamyl alcohol (24:1)
6. RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
7. Proteinase K:  10 mg/ml stock
8. 70% ethanol
9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use.

Procedure:

      1.    Transfer about 10-20 mg animal tissue to an MFT 1.5mL tube labeled with an identification         number.

      2.    Add liquid nitrogen and grind the tissue for 1 minute with a pestle.

      3.    Add 300μl Extraction buffer and 100μl SDS 5%.

      4.    Add 15μl Proteinase K.

      5.    Grind the tissue with a pestel.

      6.    Incubate the tube at 60°C for at least 30 minutes.

      7.    Add 1-2 µl of RNase and incubate for 10 min at RT.

      8.    Add equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10 min at RT.

      9.    Centrifuge at 8,000 rpm for 15 min.

     10.  Carefully decant the aqueous (top) phase to a clean microfuge tube.

     11.  Add an equal volume of chloroform: isoamyl alcohol (24:1) and mix gently and Centrifuging       at 8000 rpm for 5 min.

     12.  Decant the aqueous phase to a clean microfuge tube and add 1/10 or equal volume of 3 M          sodium acetate and 300μl 10 or equal volume of isopropanol.

      13.  Mix gently inverting the MFT.

      14.  Leave in cold temperature for 30 minutes.

      15.  Centrifuge at 13,000 rpm for 5 minutes.

      16.  Eliminate the supernatant and collect pellet (in same MFT).

      17.  Wash pellet adding 500μl ethanol 70%.

      18.  Centrifuge at 13,000 rpm for 5 minutes.

      19.  Eliminate the supernatant.

      20.  Dry the pellet for 10-15 minutes at 60°C to evaporate remaining ethanol.

      21.  Resuspend pellet in 50μl TE.
                 Downloads...

Difference between BOD and Bacteriological Incubators.

Bacteriological Incubators:

An ordinary incubator is generally based on ambient temperature for growth bacteria. It can have some basic heating arrangements. Bacteriological incubators are used to provide temperature range of 30-35°C for the growth of bacteria. Bacteriological incubators works at ambient temperature and it contains only heating mechanism. In these bacteriological incubators, if set temperature is in range of 30-35°C then they can only maintain their set temperature if surrounding environment temperature is below or in a range of 30-35°C. If surrounding temperature is higher then the incubator set temperature then there would be rise in display temperature of incubator because of high temperature of surrounding air and that air is used for the circulation in the chamber. Bacteriological incubator has no cooling mechanism to lower the display temperature. It fails in cold weather. If the room where it is stationed has right ambient temperature, it can still work.

BOD incubator:

Bod incubator has full control over the temperature as it is provided with both heating and cooling arrangements and provided +/- 0.5 0 C variation over the set temperature. It does not provide control over relative humudity. The environmental chamber provides control over temperature and relative humudity. BOD incubators are used to provide low temperature in range of 20-25°C for the growth of fungus and bacteria. BOD incubators have heating as well as cooling mechanism and they work independent of surrounding temperature. BOD incubators contains heating coils as we as compressor for cooling process. If incubator set at temperature of 20-25°C and surrounding temperature is higher than this set temperature then BOD incubator maintain the set temperature with the help of cooling mechanism and if surrounding temperature is low as compare to set temperature then heating would be generated with the help of heating coils. BOD incubators are also called low temperature incubators. These BOD incubators could be used as bacteriological incubator by changing the incubation set temperature requirements.

Mr. Khadga Bikram Angbuhang

Faculty: Microbilogy,

GoldenGate International College, Nepal

Research officer: LEAD Nepal

Past: Microbiologist, Chaudhary Group

Nepal

Determination of Dissolved Oxygen in Water by Titrimetric Method

Determination of Dissolved Oxygen in Water by Titrimetric Method

(Winkler method)

INTRODUCTION

The term Dissolved Oxygen is used to describe the amount of oxygen dissolved in a unit volume of water. It is a measure of the ability of water to sustain aquatic life. Dissolved oxygen (DO) is essential for the maintenance of healthy lakes and rivers. Any reduction in the amount of oxygen in a body of water, caused by a rise in temperature or by pollution, may mean that certain animals can no longer live there. The concentration of dissolved oxygen can therefore be taken as a measure of the ability of water to support living things. lf the oxygen concentration is less than half the maximum concentration then the water is of poor quality.........