Hi Zelalem,

Please give us some more information about your experiment.

Which pDONR vector do you use?

You may have amplified your DNA fragment by attB-PCR.

How may bp of the PCR fragment?

How do you purify the PCR product ?

Do you precisely adjust the concentration of the DNAs in the reaction?

I believe that the purity, concentration and molecular ratio of the DNAs are most important in BP reaction.

Thanks.

@ Takefumi Sone

Here is the details for your questions

I am using pDONR221 and used attB flanked PCR product which has a size of 3.4kb. I run my PCR product on the gel and purify it from the gel. I made the final concentration of my insert 50ng/ul and used 2.3ul of this (approximately 115ng). Th amount of pDONR was 300ng.    

Nothing sounds so strange, hmm....

If you have experience of successful case, we don't have to doubt the activity of your BP enzyme. Do you? If not, you should doubt the enzyme.

If you have succeeded in other case, try higher competency bacteria such as Mach1 and incubate longer time after heat shock before plating onto Km plate.