Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP)
The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it (1). Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector must be optimized. Even then, the resulting library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing random mutagenesis libraries.
KeywordsExonuclease Activity Template Plasmid Specific Restriction Enzyme Single Insert Multiple Insert
- 1.Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar