Team:Amsterdam/project/gibson assembly/
From 2012.igem.org
Gibson Assembly
Contents |
What is Gibson Assembly
The Gibson Assembly is a new way of cloning. Instead of restriction/ligation based cloning it uses specialized primer to do this job. This process has proven itself not only as innovative but also as a different way with just as much success as the classical way. And the method is up and coming in many laboratories and is getting much more used every day.
Why use Gibson Assembly
We were considering from the start which way of cloning to use since we, like any other iGEM team, have a great number of cloning to do. So we wanted to choose a reliable and fast way of getting from construct A to construct D, which Gibson Assembly suggest to do. Plus one added benefits of using Gibson Assembly is that is does not leave a scar, something that’s bound to appear when using lots of classical cloning. This is what made us decide to explore the Gibson Assembly and aim to get this working for us.
How Gibson Assembly works
As mentioned above the Gibson Assembly uses specialized primers to replace the digestion/ligation process. First both parts, the vector and the insert, need to undergo a PCR with these primers to generate a good quantity of bands that have an overlapping part, generates by the primers. The primers actually consist of the normal primer part and a tail, in which case the tail would correspond with a sequence of the other part. Using a Gibson-mix, containing polymerase, exon-nuclease and ligation material, the Gibson method is able to lay open these overlapping parts, using the exon-nuclease that eats away some bases, to bind, ligate, and repair, polymerase, all of this into one whole plasmid. Which can then be transformed into bacteria and be selected.
Gibson Assembly Protocol
Primer design
Of vital importance for the Gibson Assembly is the primer design. Normally Gibson primers are about 40 bp in length, each time 20 bp make up for the primer and the tail part. The primer part should of course work as a normal primer should. The part of the tail should correspond not with the PCR product but with the other PCR product, which it shall meet later on. This also goes visa versa for the last mentioned PCR product. Otherwise when making these primers close attention needs to be upheld to no create self-dimers, primer-dimers, hair-pins and not overshooting the Tm or GC content.
Protocol
All steps take place under one temperature, … C, in the same vail. An ratio of DNA to each other should be uphold. Otherwise the Gibson Method does not differ very much from other classical cloning. Note; see protocol page
Material/Mixes
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