Hello,

I used this kit for direct mutagenesis and my Ligation step sometimes works and sometimes doesn't. Can someone please tell me what worked for them?

Protocol:

PCR Product: 10-20ng (I usually put 3ul of the Dpn1 digested PCR)

Ligation Buffer: 2ul

Ligase: 0.5ul

H2O: 4.5ul

I have tried

PCR Product: 3ul

Ligation Buffer: 2ul

Ligase: 0.5ul

H2O: 3.5ul

ATP:1ul

PCR Product: 1.5ul

Ligation Buffer: 2ul

Ligase: 1ul

H2O: 4.5ul

ATP:1ul

My tries worked for some plasmids and didnt work at all for some. I need help with figuring out what would work for all of them so I have cohesive protocol.