User:Mbennie/Notebook/Papers and Books/Methods
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- Ko JK and Ma J. A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study. Am J Physiol Cell Physiol. 2005 Jun;288(6):C1273-8. DOI:10.1152/ajpcell.00517.2004 |
- Uses type II restriction enzymes to rapidly induce desired mutations into DNA
- Designs two primers around mutation site with type II (Ear1, BsmA1, BsmB1, Sap1, Ksp6321, BpuA1, Alw261, Eam11041) restriction enzyme sites
- Performs PCR to create two strands of DNA with mutation and restriction enzyme sites (100ng of template, 1 uM primer pair, 200 uM dNTP, 2.5 U of Taq polymerase)
- Cuts DNA with restriction enzymes and ligates back together (10 U enzyme, 400 U T4 DNA ligase)
- Performs gel purification to get desired product
- Cuts and ligates into a plasmid
