If one were to assemble such a large gene from those small fragments into a plasmid, what method would be preferable? Those fragments may be available as a part of a plasmid or as a PCR product.
The exact number of fragments isn't explicitly defined here but due to afromentioned constraints, it will be higher than 10, perhaps 12, plus a plasmid backbone.
Golden gate method and perhaps Gibson assembly seem to be some possibilities, provided that specialised protocol and products are used.
Dear Marcin Goławski
It is common to combine about 10 fragments simultaneously by restriction enzyme treatment and ligation reaction.
However, if you are not an expert in plasmid construction, it is not recommended.
On the other hand, I have introduced five 1kb DNA fragments into one plasmid vector simultaneously by restriction enzyme treatment and ligation reaction.
The method was to prepare various fragments of about 10 ng/uL, mix them in 2 μL each, mix with 2xLigation kit, and perform ligation reaction at 4°C o/n.
The ends of each fragment were designed to be unique. In other words, only the right side of the A fragment and the left side of the B fragment can bind to the C, D & E fragment.
The fragments can be easily prepared by using asymmetric restriction enzymes such as BsaI and BbsI and PCR.
For your reference.
In addition, by using the overlap PCR method, multiple fragments can be combined by the PCR method.
Alternatively, multiple fragments can be ligated to obtain a combined fragment, and then the single fragment can be mass amplified by PCR for easier introduction into the vector fragment.
Best,
Narumi
As the chance of correctly assembling a set of fragments decreases with the number of fragments, it may be advisable to proceed in more than one step, i. e. by first assembling and verifying a set of larger subfragments, before piecing these together into the complete segment.
It'd appear now that I can obtain a plasmid containing the desired sequence in a different way but I'd like to thank for the answers.
Narumi Uno May I ask you an additional question? for example, the Bbsl cloning site shows it would cut after some N bases, does that means we cannot predict the specific cloning site of Bbsl? which may lead the fragment ligase to other enzymes we don't plan to?
I am not familiar with this enzyme, thank you in advance!
Jiahui Niu The cut section of BbsI is not unpredictable, rather arbitrary cut surfaces can be fabricated.
Since BbsI recognition and cleavage sequences are denoted as GAAGAC(N)2/(N)6, etc., it is certainly not possible to define the cleavage section sequence of the naturally occurring BbsI site. However, an arbitrary 4-base 5' sticky end can be created by preparing any sequence using artificial synthetic oligos, etc.
It is difficult because I am busy today, but if necessary, I can illustrate tomorrow?
Narumi Uno Thank you for your reply. If I use bbsl to cut 2 plasmids we already have and recombine them together, the cloning site is known, and bbsl can just work like other restriction enzymes like sca1 or nco1? I am just unsure about what the (N)2/(N)6 part means.
No, it can not.
A schematic diagram is The sequence of the BbsI cleavage site depends on the DNA sequence being cleaved, so without knowing the detailed sequence, it is not possible to know if the cleaved DNA fragments by two different BbsIs can be recombined.shown with respect to cleavage and ligation at BbsI.
On the other hand, ScaI and NcoI are strictly defined in the cut section.
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