From PCR amplification, you usually get a lot of product; it should be enough to do 5-6 cloning. The concentration out of a PCR reaction is usually in micrograms, whereas you would need ng for your ligation.

Thank you so much! Because I read somewhere that they recommend using 100 ng of dna for every single part.

I don't wish to have many transformants / (CFU) anyway on my plate. Only one if correct would be great. So you really help me with your answer, I can have a little idea of what to expect

I will try not to use intermediate vector then and do the direct cloning. For the parts I will use only a few times. And for parts that I will include in more constructs, such as sfGFP, probably either do multiple PCR or clone into an intermediate vector, amplify and then digest and mix with PCR parts for ligation.

Thank you very much for your answer, it is really useful for me :)

Try this out, if you want. Works for me everytime.

https://youtu.be/9b1V2HKhg5A

Try this out, if you want. Works for me everytime.

https://youtu.be/9b1V2HKhg5A

Hi Adrian Ionut Cepleanu-Pascu ,

Thank you for the answer, and your video was very informative, I actually had the same idea as you at first but I didn't understand that it was possible to skip fragments until I watched your video.

You made me understand that for skipping a fragment N, it is simple, by just changing one primer on the fragment N-1 to have the 4 letters overhang of the fragment N+1.But also I learned about the SMAR element which I didn't know before.

That is so smart ! Thank you so much.