The transfer of denatured DNA from Agarose gel to Nitrocellulose Blotting or Filter Paper technique was introduced by Southern in 1975 and this technique is called Southern Blotting Technique.
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The transfer of DNA from agarose gel to nitrocellulose filter paper is achieved by Capillary Action. A buffer Sodium Saline Citrate (SSC) is used, in which DNA is highly soluble, it can be drawn up through the gel into the Nitrocellulose membrane By this process ss-DNA becomes 'Trapped' in the membrane matrix. This DNA is hybridized with a nucleic acid and can be detected by autoradiography. Autoradiography - A technique that captures the image formed in a photographic emulsion due to emission of light or radioactivity from a labelled component placed together with unexposed film.

Agarose Gel Electrophoresis is used mainly for the purification of specific DNA fragments. Agarose is convenient for separating DNA fragments ranging in size from a few hundred to about 20000 base pairs. Polyacrylamide is preferred for the purification of smaller DNA fragments. The gel is complex network of polymeric molecules. DNA molecule is negatively charged molecule - under an electric field DNA molecule migrates through the gel. The electrophoresis is frequently performed with marker DNA fragments of known size which allow accurate size determination of an unknown DNA molecule by interpolation. The advantages of agarose gel electrophoresis are that the DNA bands can be readily detected at high sensitivity. The bands of DNA in the gel are stained with the dye Ethidium Bromide and DNA can be detected as visible fluorescence illuminated in UV light will give orange fluorescence, which can be photographed.

A technique in which the pattern of colonies growing on a culture plate is copied. A sterile filter plate is pressed against the culture plate and then lifted. Then the filter is pressed against a second sterile culture plate. This results in the new plate being infected with cell in the same relative positions as the colonies in the original plate. Usually, the medium used in the second plate will differ from that used in the first. It may include an antibiotic or without a growth factor. In this way, transformed cells can be selected

a. Chemical mediated gene transfer: Certain chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant + protoplasts.
b. Microinjection: The DNA ""+ is directly injected into the nucleus using fine tipped glass needle or micro pipette to transform plant cells. The protoplasts are immobilised on a solid support (agarose on a microscopic slide) or held with a holding pipette under suction.
c. Electroporation Methods of Gene Transfer: A pulse of high voltage is applied to protoplasts, cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs.
d. Liposome mediated method of Gene Transfer: Liposomes the artificial phospholipid vesicles are useful in gene transfer. The gene or DNA is transferred from liposome into vacuole of plant cells. It is carried out by encapsulated DNA into the vacuole. This technique is advantageous because the liposome protects the introduced DNA from being damaged by the acidic pH and protease enzymes present in the vacuole. Liposome and tonoplast of vacuole fusion resulted in gene transfer. This process is called lipofection.
e. Biolistics: The foreign DNA is coated onto the surface of minute gold or tungsten particles (I-311m) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/micro projectile gun/shotgun). Then the bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells.

(i) Blue- White Colony Selection Method is a powerful method used for screening of recombinant plasmid.
(ii) In this method, a reporter gene lacZ is inserted in the vector.
(iii) The lacZ encodes the enzyme β-galactosidase and contains several recognition sites for restriction enzyme.
(iv) β-galactosidase breaks a synthetic substrates called X-gal (5-bromo - 4 -chloroindolyl- β-D-galactose- pyranoside) into an insoluble blue coloured product.
(v) If a foreign gene is inserted into lacZ, this gene will be inactivated. Therefore, no-blue colour will develop (white) because β-galactosidase is not synthesized due to inactivation of lacZ.
(vi)Therefore, the host cell containing r-DNA form white coloured colonies on the medium contain X-gal, whereas the other cells containing non-recombinant DNA will develop the blue coloured colonies.
(vii) On the basis of colony colour, the recombinants can be selected.