Can you please explain how the last two primers are sitting over the template strand as the restriction site is now in the middle of primers

Oh my gosh. This was so helpful. Thank you.

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What I have been looking for....thank you for the wonderful explanation

Thank you for the video. But I didn't understand why we design both forward and reverse primers on both sides. Isn't it enough if we have one forward primer matching the left side of sfGFP, with the overhang marching the backbone, and a reverse primer for the right side of sfGFP, with the overhang matching the other side of the backbone? Do you use 4 primers for the PCR?

Very good explanation. Highly recommended.

11:10 is a mistake

thanks! absolutely wonderful explanation.

Thanks..this is by far the most useful help that I got for my cloning.

Thank you for this great video Where's the music from?

this is 40bp overlap, according to the manual, should 15-25bp overlap be enough?

It was super useful to START understanding. I need more explanations. Can someone please recommend me places where I can study from? :´(

Thanks, this is very helpful !! :)

Why would retreating to the 19th base pair be prone to the primer misaligning?

if I assembled two genes together, when inserting the recombinant DNA into an expression vector, the products wouldn't be altered?

Terrific video. I had never heard of Gibson Assembly before, but after seeing this and the NEB videos, I think I could do it.

The Music is very catchy, where i can find it.

Thank you so much for the video. It is very helpful for me!

Thank you Corinna, it is the best explanation I have ever seen;-)

Hi, I am new in this therefore your help would be appreciated. So what is the next step? A PCR where you use both intact plasmid (the plasmid on the left in the video-without the insert) and the sfGFP at the same time?

quite clear, quite helpful! thank you!

HindIII produces sticky ends, how would this would work with GA?

what software do you use for drawing your plasmids?

Thanks, that was explained with great clarity. I have a question: After the primer design here, do you still need to check for formation of haripins/primer dimers? Or is this design good enough for a successful ligation using the Gibson Master Mix?

Great tutorial!