There is a recent publication titled "In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning" (J. Biol. Chem, 2019; doi: 10.1074/jbc.REV119.009109), which revisited an old topic on bacterial in vivo cloning.
Although the authors clearly illustrated the concepts and procedures to perform the experiments, there were actually no experimental data in the paper. I am wondering whether any of you have tried this method in your molecular cloning? If so, are there any tips and/or pitfalls others who have never done it (like us) should be aware of?
Thank you very much!
Hi Gang,
after using the InFusion kit, we no use routinely what we call Aqua cloning (from this paper 1. Beyer, H.M.; Gonschorek, P.; Samodelov, S.L.; Meier, M.; Weber, W.; Zurbriggen, M.D. AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach. PLOS ONE 2015, 10, e0137652.) in Xl1-blue or stellar Ecoli. We had only a handful of cloning that didn't work and we went back to the old good T4 ligase. But I recently had trouble for 1 gene with a specific internal domain that was deleted in over 50% of the clones, so full and careful sequencing is required. We also used this approach to ligate 2 inserts at once, and it works quite well.
Dear Dong
i'm routinelly using the PIPE cloning approach, which is an in vivo cloning approaches developer from NIBR center of Novartis
Article The Polymerase Incomplete Primer Extension (PIPE) Method App...
Article Combining the polymerase incomplete primer extension method ...
that a learned when i worked for Novartis Vaccines in Siena.
I used it to clone many ORF in pet15 and pcdna3.4 as well us to generate undreds of mutants, small instertion or deletion and it work very well.
The main problem is related to the E.coli strain able to perform the reaction because in my experience the most of the standard strains suitable for cloning do not work. THe best strain are the HK100, which are Novartis proprietary and not commercially available and the only commercial strain that i know that work well are the thermo mach1 which are not very cheap. However with this approach you do not need to perform PCR purificazione, digestion, ligation and therefore also if cell are expensive you save a lot of money in the other steps.
If you are interested to know more details about it you can find a video about Primer desing for PIPE cloning on my blog ProteoCool https://proteocool.blogspot.com/
recently Takara and Lucigen develope 2 kits that seems to be based on the same approach:
https://www.takarabio.com/learning-centers/cloning/in-fusion-cloning-overview
https://www.lucigen.com/Expresso-Cloning-and-Expression-System/#subcat-tabs2
i never use thos kits, but some my ex colleagues told me that the Takara kit seems to work very well.
good luck
Manuele
Check out the following seamless restriction ligation free cloning method
https://immunolabsolutions.com/2019/06/26/seamless-restriction-free-cloning/
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