Colony PCR running as smears - (Jul/25/2007 )
Can anyone please help I have wasted 4 days monkeying around with this.
I have tried colony PCR using fresh colonies of a transformation. I have tried different methods. I have tried to pick a colony, put it in water, heat 95C for 10:00, spin in a microfuge and use 1 ul as template. I have tried doing the same procedure without spinning the lysate. I hate tried adding a small amount of colony to a 1X PCR mix and doing a 5 min initial denaturation, No matter what the result is always A BIG FAT SMEAR. Please help.
Are you certain the PCR reaction itself is working? That is, can you get a sharp band from other template DNA and the same primers? If not, that is the first step. A common problem is too many cells in the PCR reaction. Try diluting your cell suspension by 10x and 100x.