Ligation Independent Cloning

The well established restriction enzyme cloning method, i.e. looking for a (unique) restriction site, cutting out a fragment and inserting another one with corresponding sites, ligate the two fragments and then transform it into bacteria, has a few limitations. First, having the correct restriction sites at the perfect place is not always happening. Second, some of your inserts or fragments carry that restriction site as well, all you will do is just chopping your insert into pieces. Fortunately, there’s another great option: Ligation-independent cloning. All you do is putting two fragments with some homology together, add some fancy proteins and voila, it’s done. Here, I will mention two great system: Gibson assembly and SLiCE.

Gibson Assembly (GA)

Back in 2009, Daniel Gibson and collegues published a wonderful paper about cloning hundreds of kilobases in a one-pot reaction. The idea is great and the system easy to understand.

gibson assembly scheme

Basically, in the master mix, what you either can create yourself or buy for example @ NEB, there is T5 Exonuclease (Step B, chewing back 5′ ends), Phusion polymerase (Step D, refilling) and Taq Ligase (Step E, ligation) included. The enzymes are stable at a relatively high temperature and thus, the reaction can be performed at 50°C. For two or three fragments (like in the example above), you normally incubate 15 min, for more fragments and/or low efficiency you may incubate the reaction for 60 min.

The commercial product comes as a 2x MasterMix and is around 159 USD list price for 10 rxn, making it not very cheap per reaction (16 USD). However, I am normally going with a 10 µl rxn instead of 20 µl and get similar results. And I figured out that Gibson assembly is quite robust and deals very well with low concentrations and unpurified PCR products.

Tipps:

  • The reaction performs at 50°C, therefore the homology ends should anneal at this temperature. Otherwise it results in very low efficiency.
  • Do half the reaction volume to save money

Seamless Ligation Cloning Extract (SLiCE)

Accidentially found, SLiCE is a simple, very cost-effective bacteria cell extract containing proteins mediating a GA-like reaction not depending on the “known” RecA system. Published three years ago in Nucleic Acid Research, a lot of labs transitioned to that great and inexpensive system.

You prepare your backbone and your inserts similar to GA, however, the reaction performs at 37°C, meaning that the homology arms have not that 50°C melting temperature restriction. Notably, when using PPY bacteria for generating the SLiCE, you only need 15 bp to have a relatively high cloning efficiency and 99% accuracy. However, 20-30 bp seem to be more efficient (see Table 1).

A very convenient protocol that works for a lot of people:

1 µl 10x T4 Ligase buffer (contains already ATP; fresh aliquot – do not freeze and thaw)
1 µl Backbone DNA (PCR amplicons have significantly lower efficiency, ca. 100 ng in total)
3 µl Insert DNA (e.g. purified PCR amplicon, ca. 300 ng in total)
4 µl ddH2O
1 µl SLiCE (aliquotted)
10 µl TOTAL

Incubate the reaction for 37°C 15-60 min depending on your fragments.
You may troubleshoot the best transformation protocol, the original publication used 1 µl in 100 µl of bacteria; I am pretty stingy, thus, I am using 1-2 µl in 20 µl bacteria. Update: Actually, now I did my own bacteria (stay tuned for another post) and use 1.5 µl in 100 µl of DH5alpha cells. SLiCE reaction seems to be highly effecient for 2-3 fragments with 30 min incubation.

Tip: Be careful with gel extracted DNA! SLiCE seems to be very susceptible for UV-treated DNA. Thus, I am cutting the gel blindly without exposing it to any UV light.

Another great feature of the SLiCE method is that it works with heterologous arms. For example, you have a vector harboring GFP and you would like to replace it with mCherry. However, there are no restriction sites around GFP and your beloved homology arms are buried inside of the vector. The solution is just cut the vector between your desired homology arms, do your PCR with the homology arms and do your SLiCE. It is capable of removing the heterologous DNA and combining the homology arms to achieve the desired end product. See this figure for clarity.

If you have any questions, please use the comments box below.