How to design primers for Gibson assembly

[@roomiemcgee8899]

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

[@mahasish]

Very good explanation. Highly recommended.

[@soumyadipXplore]

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

[@johnoladokun6230]

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

[@djdonald2795]

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

[@alexcracker]

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

[@AmandeepSharmaP24BB000]

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

[@ranjit2289]

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?

[@nupur0003]

thanks! absolutely wonderful explanation.

[@poiuytrew09876]

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?

[@poiuytrew09876]

What I was meaning was, do we really need primers for the vector? When we digest them with restriction endonuclease, it's already linear.

[@polasoliman3955]

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

[@andrevargasaguilar2723]

because yes.

[@aBirdbyBrancusi]

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

[@noahyi1903]

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

[@skaplan1995]

Great tutorial!

[@cathytang2558]

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

[@kopolojoyono]

quite clear, quite helpful! thank you!

[@ArshadPadhiar]

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

[@strabana5806]

Thanks, this is very helpful !! :)

[@noadrow]

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

[@ismailcan5635]

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?

[@MrAntihumanism]

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

[@rhlsy3209]

I think you would have to switch enzymes to something that produces blunt ends

[@Trypanosoma_]

HindIII is not being used at all. A site for it was incorporated into the primers but no enzyme is being introduced

[@vivekdna1]

what software do you use for drawing your plasmids?

[@jakehay9406]

Vivek Kumar pretty sure its SnapGene

[@vivekdna1]

Thanks Jake

[@Dianax.]

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

[@jamhaslam612]

if you wanted the two genes to be expressed as two separate proteins you would need either an IRES site or T2A in between the two genes.

[@zilog-q3u]

11:10 is a mistake

[@SohaMokhtariGarakani]

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