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RDNA (recombinant DNA technology) is a artificial method that is used to obtain the clones. It is obtained by combining two or more sequences that undergo gene splicing .It begins with the isolation of gene and then inserted into a vector for cloning procedure. This method is used in various different areas such as prophylaxis, diagnosis, therapy etc. In this experiment, we use donor DNA and vector DNA for restriction process and then ligated into a vector, which then be transformed into a bacterial host cell.
Recombinant DNA (rDNA) is a method that is used to combine the DNA segments. This rDNA molecule is formed by merging two or more different DNA molecules. It is an artificial technology involved in gene cloning method. It is a field of molecular biology in which we form new synthetic molecules commonly called as chimeras. This technology plays a major role in scientific research and also in diagnosis, genetic disorders & in many areas of medicine.This method will undergo in four stages to form clones of bacteria Isolation restriction, ligation ,bacterial transformation.
This method mainly involved in isolation of plasmid DNA from the bacterial genome. We need to first extract and purify DNA and then analyse it by gel electrophoresis method. Isolation method consists of two parts, one it will isolate the DNA by fragmentation process and other is to separate the plasmid. This ligation procedure is done in two different methods for isolating plasmid DNA Alkaline lysis ,QIAprep spin plasmid kit
The enzymes that are used to cut the DNA molecules are called restriction enzymes. In this restriction endonuclease analysis, we use phase lambda and pUC19 that are cut with these enzymes. The obtained results are then analysed by agarose gel electrophoresis. Restriction endonuclease are the enzymes that are used to cut the inter diester bonds at different restriction sites to give the primer ends of the DNA molecule with blunt or sticky ends.
Restriction undergo in both DNA and RNA at different sites by restriction enzymes that includes Ecor1, Sac1, BamH1, Hind111 and so on. In our experiment we used EcoR1 endonuclease enzyme to cut the DNA molecule by removing the phosphodiester bonds.
In order to clone the DNA we first need to cut the edges by using restriction endonuclease enzymes. we know insert the DNA fragments into the vector molecule.DNA ligase enzyme is used to combine the two different DNA fragments. Ligation starts from the 5' phosphate group to the 3' hydroxyl group. Ligation reaction works with the cohesive ends but it is not used for blunt ends. This ligation step undergo in all the bacterial organisms.DNA fragments are inserted into the DNA by T4 DNA that combines the 3'OH and 5'phosphoryl ends by using Mg2+ and ATP as a co-factors. In this ligation, T4DNA ligated the lambda DNA and pUC19 that circularise at the end of the ligation analysis.
It is the basic step in the inviter analysis that shows the DNA into the living cells. Transformation is that transforming the naked DNA into the host cells. The obtained cells get transformed and that contains the copies of the DNA. The most common method we used in this experiment is that involving high calcium concentration and heat. Transformation step was first observed by F.Griffith in which genetic information is sent by means of the extracellular spaces. It is used for building up of the genetic maps in bacterial cells .Its main work is to form colonies of the E.coli that handling the lambda genome that are to be inserted into the pUC19
The main aim is to form the clones of E.coli having the portions of the lambda genome that puts into a plasmid pUC19 by using restriction, ligation, transformation, isolation methods.
Materials and Methods:
The overall procedure of this restriction, ligation, transformation, isolation experiment was followed as per core molecular biology practical booklet.
(this gel picture has been referred from the batch group of mohan,as I was on leave on the particular lab session).
WELL 1 = DH5Î±
WELL 2 = pUC19
WELL 3 = Î»DNA + ECORI ( sample 1)
WELL 4 = uncut pUC19 (sample 2)
WELL 5 = pUC19 + ECORI (sample 3)
Distance travelled by Î» DNA ( mm)
DH5Î± + uncut pUC19
Cut pUC19 +
Cut Î» DNA
Cut Î» DNA
DH5 Î± +
Cut Î» DNA
Percentage of Transmission = (Total number of white colonies in plate 4 and 6 / total
Number of colonies ) x 100
= (5/18) x 100
= 27.77 %
Efficiency of transmission = (total number of colonies growing on plate 4 and 6/ Amount
of DNA spread on agar plate (in µg)) x Dilution factor
= (18/0.5) x10
= 360 cfµ of DNA
In this experiment, we observed that Î»DNA and plasmid pUC19 were precised with EcoR1.We have observed totally six fragments in the above gel picture.In the first lane we have obtained light bands,and the length is maximum out of all fragments.The third and the fourth one are combined.