Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.
Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. If the PCR yield is low, more product can be added to the KLD reaction, however a buffer exchange step, such as PCR purification, must be included prior to transformation.
Use NEBaseChanger™ to design primers and calculate annealing temperature
Troubleshooting tips for Q5 Site-Directed Mutagenesis Kit