Properties of Good Host

 A good host have the following features:-

1. Should be easy transform,                                                                                                          
2. Should support the replication of recombinant DNA,                                                                     
3. Should be free from elements that interfere with replication of recombinant DNA,                         
4. Lack active restriction enzyme, e.g. E.coli K12 sub strain HB 101,                                   
5. Should not have Methylases since these enzyme would methylate the replicated recombinant DNA which, as a result, would become resistant to useful restriction enzymes,                                                                                                                              
6. Should be deficient in normal recombination function so that the DNA insert is not altered by recombination events.                                                                                                                                                                                                                                     E.coli support several type of  vectors, some natural, constructed, which can be grouped as follows:-                                                                                                                                                                                                                                                    1. Plasmid                                                                                                                          2. Bacteriophages (both natural)                                                                                      3. Cosmids,                                                                                                                        4. Phasmids                                                                                                                      5. Shuttle vectors,                                                                                                            6. Artificial chromosomes                                                                                                  7. Phagemids,                                

Types of Restriction Endonucleases

There are the following four distinct types of restriction endonucleases:-

Type 1st restriction endonucleases:- are complex endonucleases, and have recognition sequences of about 15 bp; they cleave the DNA about 1000 bp away from the 5'-end of the sequence "TCA" located within the recognition site, e.g, EcoK, EcoB, etc.

Type 2nd restriction endonucleases:- are remarkably stable and induce cleavage either, in most cases, than 350 different type 2nd endonucleases with over 100 different recognition sequences are known. They require Mg⁺² ions for cleavage. The first type 2nd enzyme to be isolated was HindII in 1970. Only type 2nd restriction endonucleases are used for restriction mapping and gene cloning in view of their cleavage only at specific sites.

Type 3rd restriction endonucleases:- are intermediate between the type 1st and 2nd enzymes; they cleave DNA in the immediate vicinity of their recognition sites, e.g, EcoP1, EcoP15, HindIII, ect. Type 1st and 3rd restriction enzyme are not used in gene cloning. The type 3rd enzymes recognize asymmetric target sites, and cleave the DNA duplex on one side of the recognition sequence up to 20 bp away.

Steps in GENE CLONING

Steps in Gene Cloning:-

The entire procedure of gene cloning or recombinant DNA technology may be classified into the following five steps the convenience in description and on the basis of the chief activity performed.

1. Production and isolation of the DNA fragments to cloned.                     
2. Insertion of the isolated gene in a suitable vector to obtain recombinant DNA.                                                                                  
3. Introduction of the recombinant DNA into a suitable organism/cell usually we used E. coli  called host(transformation).                                                                                 
4. Selection of the transformed host cells, and identification of the clone containing the desired gene/DNA fragment.                                   
5. Multiplication/expression of the introduced gene in the host.                    
6. Where needed, transfer and expression of the gene in the another organism.                                                                                                                                   

Autoimmune Disease

 Autoimmune Disease:-                                                                                       The immune system normally produces antibodies against g foreign proteins but not against the native proteins of body, that is, the immune system can distinguish between "self" and "non-self", However, in rare cases, individuals begin to produce antibodies against their own antigens. These antibodies are called autoantibodies and the diseases resulting from their presence are the autoimmune diseases. Among these diseases are paroxysmal cold haemoglobinuria, myasthenia gravis and systemic lupus erythematous. 

structure of antibody

 Structure of antibody:-

What is sterilization and Types of sterilization

 Sterilization:-

All the materials, e.g., vessels, instruments, medium, plant material, etc., used in culture work must be freed from microbes. This is achieved by one of the following approaches:

1. Dry heat,

2. Flame sterilization,

3. Autoclaving,

4. Filter sterilization,

5. Wiping with 70% ethanol,

6. Surface sterilization,

                                                            1.Dry Heat

Glassware and Teflon plastic ware, and instruments may be sterilized by dry heat an oven at 160-180°C for 3 hours. But most workers prefer to autoclave glassware and plastic ware etc. And flame sterilize instruments like forceps, etc. More recently, glass bead sterilizers (300°C) are being employed for the sterilization of forceps, scalpels, etc. these devices use dry heat.

                                     

                                                2.Flame Sterilization

Instrumental like forceps, scalpels, needles, etc. Are ordinary flame sterilized by dipping them in 95% alcohol followed by flaming. These instruments are repeatedly sterilized during the operation to avoid contamination. It is customary to flame the mouth of culture vessels prior to inoculation.

                                                  3.Autoclaving

Culture vessels, etc. Are generally sterilized by heating in an autoclave or a pressure cooker to 121°C at 15p.s.i. (pounds per square inch 1.06kg/cm²) for 15 to 40 minutes, The time being longer for larger medium volumes. Sterilization during autoclaving depends mainly on temperature. Certain types of plastic ware and some instruments, e.g., micropipettes, etc. Are also autoclave. Care should be taken to properly stopper all the vessels and to open the autoclave only its pressure gauge indicates zero pressure. 

                                                4. Filter Sterilization

Some growth regulators, e.g., GA3, zeatin, ABA (abscisic acid), urea, certain vitamins, and enzymes are heat labile. Such compounds are filter sterilized by passing their solution through a membrane filter of 0.45μ or lower pore size. The membrane filter is held in a suitable assembly, the  assembly together with the filter is sterilized by autoclaving before use. Filter sterilized heat labile compounds are added to autoclaved and cooled media, in case of agar medium, they are added when the medium has cooled to about 40°C and is still liquefied.

Laminar flow cabinets are used to create an aseptic working space by blowing filter-sterilized air through an enclosed space. The air is first filtered through a coarse profiteer to remove large particles, it is then passed through a HEPA (high efficiency particulate air) filter, which filters out all particles larger than 0.3μm. This sterilized air blows through the cabinet at 1.8 km/hr. which is sufficient to keep the enclosed working area aseptic. 

        

                               5. Wiping with 70% Ethanol 

The surfaces that can not be sterilized by other techniques, e.g., platform of the laminar flow cabinet, hands of the operator, are sterilized by wiping them thoroughly with 70% ethyl alcohol is allowed to dry.

                                    6. Surface Sterilization

 All the plant materials to be used for culture are treated with an appropriate sterilizing agent to inactive the microbes present on their surface, This is called surface sterilization. Surface sterilization protocol will depend mainly on the source and the type of tissue of the explant is the excised piece of tissue or organ used for culture.

The sterilizing agent used for surface disinfection are calcium hypochlorite (9-10%), sodium hypochlorite (2%), mercuric chloride (0.1-1%), silver nitrate (1%), Bromine water (1-2%), H2O2 (10-12%) and antibiotics (4-50 mg/l). Of these, calcium or sodium hypochlorite and HgCl2 (satisfactory results) are the most commonly used. The duration of treatment varies form 15-30min. Since these agents are also toxic to plant tissues, the duration and the concentration used should be such as to cause minimum tissue death, and the rinsing after treatment should remove them as completely as possible.

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