The next step after PCR Cleanup on the CAII DNA that was amplified, DpnI treatment was performed because the template vector from PCR had to be destroyed so it does not carry over to transformation. The was necessary because the accepting vector and the template plasmid from PCR have the same bacterial resistance gene. If both of them have the same resistance gene then bacteria that did not transform correctly would not be killed off due to the presence of double resistance. DpnI cuts the remaining template molecules after PCR reaction with selectivity based for methylated DNA from the bacteria. I used 2 ul of DpnI, 15 ul of water, 5ul of NEBuffer 4 and allow it to incubate for 37 degrees Celsius 30 min and heat inactivate at 85 degrees Celsius for 20 min. Once Again, DNA concentration was lessen due to a second PCR Clean-up.
Filed under: Uncategorized |