PlasmidsCharacteristics of plasmids
Effect of plasmid size on transformation efficiency
The goal of the ligation reaction is to insert the foreign DNA into the vector. An unwanted ligation product also occurs - religated vector. One way to minimize this event is to treat the vector with phosphatase. This removes the terminal phosphate group from the restriction site of the vector and theoretically prevents religation of the two ends. In reality though, this treatment is never 100%, so a low level of religation does occur. Thus after transformation you will have two types of cells: those which contain the original plasmid and those that contain a plasmid containing foreign DNA.
Plasmids are designed to distinguish the two types of transformation products. pBR322, the first widely used vector, utilizes differential antibiotic screening to distinguish the two types of transformation products. Let's say that we clone into the BamHI site of the vector. The insert DNA will then split the gene responsible for tetracycline resistance. But at the same time, the gene for ampicillin resistance is left intact. Transformed cells are first grown on bacterial plates containing ampicillin. This will kill all the cells that do not contain a plasmid. But we still cannot say which cells contain foreign DNA. Those cells that grew on ampicillin are then replica plated on plates with ampicillin and tetracycline. Those cells which grow in the presence of the ampicillin, but die under tetracycline selection contain plasmids which have foreign DNA inserts.
pBR322 was a breakthrough for molecular biology, but the double screening procedure was time consuming and could be subject to error. In 1981, a new series of plasmids were developed that permitted the identification of the foreign DNA containing cells in a single screening step. These are called the pUC plasmids. As with pBR322, ampicillin resistance is used as one selectable marker. The second marker is based on insertional inactivation of the E. coli lacZ gene. The wild type gene can hydrolyze a specific dye [X-Gal (5-bromo-4-indoyl-B-D- galactopyranoside)] to a blue color, and the bacterial colony is stained blue. A multiple cloning site has been inserted into this gene. This site will accept fragments ending in a number of different restriction enzymes. Upon insertion of DNA into this site, the activity of the gene is eliminated and the colony appears white in color. Thus transformed colonies containing plasmids with inserts can be distinguished from those with plasmids without inserts based on the color of the colony and the ability of the colony to grow on an ampicillin containing media.
cDNA Cloning (Cloning Eukaryotic mRNA)
cDNA cloning is a method of obtaining a DNA copy of the mRNAs that are expressed at a specific stage in the development of the plant. In this manner, you can enrich the library that you will screen for those sequences in which you are interested. The reagent required for this type of cloning approach is mRNA. These mRNAs have a poly A+ tail. This tail permits the isolation of poly A+ mRNA by using either oligo-dT or oligo-U columns. Total RNA is run through one of these columns under conditions which favor the binding of the tail to the matrix on the column. After the column is extensively washed, the conditions are changed and the bound mRNA is isolated. This is the starting reagent for cDNA cloning.
Steps in cDNA Cloning