There are three reasons why the number of white clones is so much lower than the
ID: 58838 • Letter: T
Question
There are three reasons why the number of white clones is so much lower than the number of blue clones from Bench 1 ligation reaction. The first reason is because the vector will religate to itself very efficiently in the absence of alkaline phosphatase treatment. Give two additional reasons why the number of white clones is much lower than the number of blue clones from the Bench 1 ligation reaction?
No alkaline phosphatase was used in the experiment.
Hint given: Look at the ligation efficiencies of the vector only and vector-insert reactions. Secondly, look at the sizes of the respective plasmids (2.8kb, 3.3 kb, 4.7 kb, 5.0kb, 7.0 kb, 9.0 kb, 12.0 kb, 25.8 kb) (vectors Vs recombinants).
Explanation / Answer
pUC18 is a small (2686 bp) Escherichia coli plasmid with high copy number. It has a marker gene for antibiotic resistance, complete gene sequence for beta galactosidase enzyme, which also houses the multiple cloning site. This region contains restriction sites for various restriction enzymes including HindIII. A cut in this region interrupts the gene for beta-galactosidase. The enzyme hydrolyzes lactose into glucose and galactose. It can also hydrolyze a colorless substrate, X-gal, into blue colored product. Thus, the vector itself, when transformed into E. coli and grown on a media containing the substrate, X-gal, the colonies appear blue, due to beta-galactosidase activity. However, when foreign DNA, like lambda DNA, is inserted in this vector, it disrupts the gene, as a result, beta galactosidase is not expressed and the bacterial colonies containing recombinants, appear white.
In the present bench 1 ligation reaction, the number of white recombinants are much lower than the blue vectors. There are 3 main reasons for the result. One being the self ligation of the vector in the absence of alkaline phosphatase. The other two reasons hint towards the ligation efficiency and size of lambda DNA inserts and the vector DNA.
T4 DNA ligase is most commonly used in cloning. The ligation efficiency of the this enzyme decreases with increase in the size of DNA fragments. Hence, smaller fragments of DNA with sticky ends are ligated more efficiently rather than the larger DNA fragments. This causes a decrease in the number of recombinants formed from HindIII cut lambda DNA and HindIII cut pUC18 plasmid vector.
pUC18 is a small cloning vector with the size of 2686 bp. Generally, it is difficult to incorporate foreign DNA fragments larger than the vector DNA. So, the maximum size of the vector-insert recombinants would attain about 5000 bp or 5 kb. Thus the recombinants with larger size (>5 kb; 7.0 kb, 9.0 kb, 12.0 kb, 25.8 kb) are highly unlikely to be formed. And recombinants with the size similar or smaller than 5 kb (2.8kb, 3.3 kb, 4.7 kb, 5.0kb), can be obtained more efficiently.
Collectively, the absence of alkaline phosphatase, ligation efficiency and the size of vector as well as Lambda DNA/HindIII fragments, the number of white clones (the recombinants) were far lower than the blue clones (the pure vector).
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