If you mix two of double-stranded DNA fragments with the same sticky ends from different sources e. When a recombinant vector with its foreign DNA insert gets into a host cell, it can replicate many copies of itself, enough in fact for easy isolation and study.
They can be cut with a restriction enzyme at a suitable location, leaving those sticky ends. Therefore, it will be necessary to attach linkers to either end of the ds cDNAs. Steps in the preparation of vector and ds cDNA for recombination are shown below. To prepare for recombination, a plasmid vector is digested with a restriction enzyme to open the DNA circle. Linkers are short, synthetic double-stranded DNA oligomers containing restriction sites recognized and cut by the same restriction enzyme as the plasmid.
Once the linkers are attached to the ends of the plasmid DNAs, they are digested with the appropriate restriction enzyme. This leaves both the ds cDNAs and the plasmid vectors with complementary sticky ends. The next step is to mix the cut plasmids with the digested linker-cDNAs in just the right proportions so that the most of the cDNA linker ends will anneal form Hbonds with the most of the sticky plasmid ends.
In early cloning experiments, an important consideration was to generate plasmids with only one copy of a given cDNA insert, rather than lots of re-ligated plasmids with no inserts or lots of plasmids with multiple inserts. Using betterengineered vector and linker combinations, this issue became less important. They are added to E. Recall that transformation as defined by Griffith is bacterial uptake of foreign DNA leading to a genetic change. The transforming principle in cloning is the recombinant plasmid!
The transformation step is shown below. So when the recombinant cells are plated on agar, how do you tell which of the colonies that grow came from cells that took up a recombinant plasmid? Both the host strain of E. One such plasmid vector carries an antibiotic resistance gene.
In this case, ampicillin-sensitive cells would be transformed with recombinant plasmids containing the resistance gene. When these cells are plated on media containing ampicillin a form of penicillin , they grow, as illustrated below.
Untransformed cells cells that failed to take up a plasmid lack the ampicillin resistance gene and thus, do not grow on ampicillin-medium. But, there is still a question. How can you tell whether the cells that grew were transformed by a recombinant plasmid containing a cDNA insert?
It is possible that some of the transformants contain only non-recombinant plasmids that still have the ampicillin resistance gene!
To address this question, plasmids were further engineered with a streptomycin resistance gene. For example, a root cDNA library will be expected to have many clones of genes that would not be found in a leaf cDNA library. These would be clones of genes that are expressed in all cells in the plant. Genes are expressed at different levels in cells.
When the mRNA sample is used to make a cDNA library, the library will contain proportionally more copies of clones of some genes compared to others. A genomic library could have a clone of every gene for that organism. Bacteriophage vectors possess the following advantageous over plasmid vectors: Are more desirable when a large number of recombinants are required for cloning low-abundant mRNAs as recombinant phages are produced by in vitro packaging. Can easily handle and store large numbers of phage clones, as compared to the bacterial colonies carrying plasmids.
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