So I'm new to Gibson Assembly. I have done restriction enzyme ligation before. As I understand, Gibson Assembly inserts a gene of interest into a the backbone of the vector primer by having the forward and reverse primers of the vector overlap with the forward and reverse primers of the insert, inserting a gene insert into amplified vector backbone which includes the sequence of the vector around where the beginning and end of the insertion site are via overhangs on the 5' or 3' end of the primers.
My convention is denote the saved fasta sequence of strands of my vector and insert as the plus "+" strand which has forward primers that go around in the clockwise direction, and the reverse complement as the minus "-" strand which has reverse primers that go in the counter clockwise direction.
My coworker suggests that I insert a gene of interest into my plasmid like this:
Forward (+) primer of vector:
5' - (overhang includes end of insert sequence) - (begins along vector at the desired end site for insertion) - 3'
Reverse (-) primer of insert:
Simply the reverse complement of forward primer for the vector
with the same overhang, but on the 3' end.
Forward (+) primer of insert:
5' - ( along vector includes the intended beginning of the insertion site) - (overhang includes beginning of insert sequence) - 3'
Reverse (-) primer of vector:
Simply the reverse complement of forward primer for the insert, except the same overhang is on the 5' end of this primer.
This simplifies primer design. The overhangs of the primers match up perfectly. So if I know the forward primer of the vector then I know the reverse primer of the insert.
However, I'm concerned that this method will cause the primers to anneal together, inhibiting their attachment to the vector and the insert.
My vector plasmid is much bigger than the insert, so I think I should amplify my vector around the desired insertion site, but not put overhangs on these primers for the vector.
Then I will design the insert primers with overhangs that match the vector like so:
Insert forward primer:
5' - (overhang includes vector sequence near the beginning of the desired insertion site) - (includes beginning of insert sequence) - 3'
Insert reverse primer:
5' - (overhang includes vector sequence near the end of the desired insertion site) - (includes the end of the insert sequence) - 3'
In this way only the insert has overhangs on the 5' end which match up with the sequence of the vector along the desired beginning and end of insertion site. So the primers should not pair up so easily and be more likely to attach to the vector and insert.
Then if I use about 5 - 6 times the amount of insert as plasmid vector then I think that should increase the probability that my gene is inserted into my vector.
What do you think?
Hello,
I think you might be overthinking this a little bit. I suggest to use gibson assembly design programs (some are freely available like NEB builder https://nebuilder.neb.com/#!/, while if you want a more advanced solution you can use snapgene, which has a free trial). You are correct that you do not need to add an overhang to your vector fragment as the entire overhang can be added to the insert (what is usually done is the that the overhang is split between adjacent fragments, this helps reduce the length of primers required for amplification of the gibson fragments). However, make sure that the annealing temperature of the overlap region is above 50°C to ensure strong binding during the assembly.
Best of luck!
Hello,
I think you might be overthinking this a little bit. I suggest to use gibson assembly design programs (some are freely available like NEB builder https://nebuilder.neb.com/#!/, while if you want a more advanced solution you can use snapgene, which has a free trial). You are correct that you do not need to add an overhang to your vector fragment as the entire overhang can be added to the insert (what is usually done is the that the overhang is split between adjacent fragments, this helps reduce the length of primers required for amplification of the gibson fragments). However, make sure that the annealing temperature of the overlap region is above 50°C to ensure strong binding during the assembly.
Best of luck!
I am using SnapGene (https://www.snapgene.com). you can have a 30-day free trial of the full program and design as much as you can during that period.
and than buy it if you liked it.
SnapGene is the best tool for every type of molecular simulations like Gibson Assembly, Gateway cloning, In-Fusion cloning, insilico PCR and all you wish to do.
It is a 30 day trial program but it will can be extended for one more month on request.
What the heck is all those things you write. Generaly use some cloning program. Copy sequence and then use NEB BUILDER TOOL.Also follow the protocol to the letter use the NEB HIFI SERIES which is an gibson assembly that can excise un paired nucleotides and is advertised as more robust.
I would say that it works. As for primers the primers and overhangs followa similar rules no too much Gc content not runs of Gs etc.
Also some thing completely understated is that the GA is not totally sequence independent. In theory yeah but, In reality you can't clone easily around structure dense sequence so choose primers accordingly
Hanna Alalam Can you explain the following part of your answer?
"you do not need to add an overhang to your vector fragment as the entire overhang can be added to the insert (what is usually done is the that the overhang is split between adjacent fragments, this helps reduce the length of primers required for amplification of the gibson fragments)"
Thank you!
Yasha N. B. if you are linearizing the vector by PCR, it mean you do not have to add gibson overhangs to the the vector amplification products (for example if the homology arm is 20 bp then it is split between adjacent fragments 10bp added to the primer of fragment 1 and 10 bp added to the primer of fragment 2) since in this case the second fragment is the vector itself you can add the entire 20bp just to the fragment to be assembled (there is an option to design fragments like this in snapgene by ticking the box that says amplify vector without extensions)
Hanna Alalam Is there any picture or a link/video that dhows us how to do this? Thanks!
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