MICRO ISOLATION, RESTRICTION AND LIGATION AND TRANSFORMATION
To isolate plasmid DNA from bacterial cells
To learn the basic procedures involved in Genetic engineering to be precise recombinant DNA by obtaining clones of Escherichia. coli [E. coli] carrying portions of the lambda genome inserted into the plasmid pUC19.
Genetic engineering's end point is to have daughter cells with an extra segment of DNA, where in the new DNA segment is introduced to the parent cell in such a way that replication and transmission is attained. This involves the isolation of the DNA sequence, creation of an artificial recombinant DNA molecule [restriction and ligation process] and introducing it into a host cell [transformation]. In the recombination experiment the plasmid utilized is that of the pUC series because, in each bacterial cell a vast amount of identical copies of the plasmid may be present, as well as due to the single-step selection of recombinant using the lac Z gene for easy indication of transformation
Isolation of plasmid DNA is useful for several molecular studies; these are used because they are circular DNA molecules capable of autonomous replication with in a bacterial cell. For isolation the most commonly used small scale method is the Alkaline lysis method. This used for large numbers of individual bacterial isolates which is important in cloning. Alkaline lysis was first described by Birnboim and Doly in 1979, it comprises of cell lysis, neutralization, cleaning and concentration of extract with the use of an alkaline solution. In the lysis stage the cell membrane is solubilized, the cell wall is disrupted, proteins are denatured and the double stranded DNA, genomic DNA as well as the plasmid are converted into single strands by disrupting the hydrogen bonds between the bases. In the neutralization step the hydrogen bonds are reestablished making it possible for the plasmid DNA to re-nature [ the reformed plasmid is soluble] however the genomic DNA are too large to re-nature making it to combine with the denatured proteins to form precipitates; Thus making it possible to isolate only the plasmid DNA via centrifugation.
In the restriction and ligation process restriction endonuclease enzymes and DNA ligases are used respectively. Restriction enzymes recognize short and specific nucleotide sequences on a double stranded DNA which is known as restriction site where they cut the DNA to produce linear fragment. DNA ligase on the other hand binds two DNA linear fragments together by catalyzing the formation of a phosphosdiester bond between the 5ʹ end of one fragment and the 3ʹ end of the other. In this experiment both the lambda and the pUC are restricted by EcoR1. The products of restriction process are thus used to form a recombinant molecule via ligation. Which in turn are used to transform bacterial cells in this case Escherichia. coli . at the end of transformation the transformants care easily identified by the insertional inactivation of the lacZ gene.
Escherichia. coli has the ability to take up DNA thus is used as the host cell for transformation experiments. in this experiement the DH5α strain was used because it lacks the ability to destroy the incoming DNA , as well as being a recombination deficient thus cannot recombine with itself or other plasmids. Thus making it a good host because it is only when these features are present in a host cell that transformation can be observed or successful.
1. Escherichia coli strain DH5α
2. 50mM glucose, 2RmM Tri-HCL, pH 8.0, 10mM EDTA containing 4 mg cm‾3 lysozyme
3. 0.2 M NaOH, 1Ph 4.8
4. Agarose gel
5. Stopping mix
The experiment was performed as indicated in the laboratory manual. However due to time constriction the Restriction process was performed first till step 5 and was later continued, after performing the alkaline lysis part of the experiment.
In step 7 of transformation to obtain a 1 in 10 dilution from tubes 2 and 4, an aliquot of 100microlitres of the sample was collected and placed in eppendorf tubes labeled 2a and 4a respectively and 900microlitres of LB broth was pipetted into each.
Gel loading and Running [ 1-7 repesensent the wells ]
1. DH5α extract
2. DH5α (pUC19) extract
3. pUC19 (+) EcoR1
4. pUC19 (-) EcoR1
5. Lambda(+) EcoR1
6. Lambda + pUC19 - ligase
7. Lambda + pUC19 - ligase
Allison L. A. (2007). Fundamental Molecular Biology. Blackwell publishing. Oxford.
Reed R., et al . (2007). Practical skills in Biomolecular Sciences. Benjamin Cummings publishing
Winter P.C., Hickey G. I., and Fletcher H.L. (1998). Instant Notes in Genetics. Bios Scentific Publishers limited, Oxford.