Dear all,
I lately tried to establish "FastCloning" in our working group, which failed badly.
The original publication can be found here: http://www.biomedcentral.com/1472-6750/11/92
I find the methodology to be highly attractive, being able to clone any sequence at any position of any vector via PCR.
However, I just cannot get the method to work - does anyone have experience in using the method and could give advice on primer design and critical steps?
In detail, I observe vector and insert amplificates of correct lengths on DNA-gel, but after Dpn1-digest and subsequent transformation, there is not a single colony obtained... I used normal DH5alpha-cells (no colonies) and high-efficiency-DH5alpha-cells (only false positives, unaltered template vector).
Any help would be highly appreciated!
Best regards,
Peter
Hi Peter,
I think this method is quite similar to overlap extension PCR cloning. I have tested the attached protocol and it worked quite well for me. I hope you find it helpful. Besides, I use these bacteria: https://www.neb.com/products/c3019-neb-10-beta-competent-e-coli-high-efficiency . Their efficiency is really high.
Regards,
Jorge
Thanks for the protocol, it looks very promising at first sight! Will give it a try :)
Have you tried Gibson assembly? A little bit expensive, but works like a charm. Plus, you can buy enzymes separately, make your own mix. Works as well as NEB's mix but cheaper.
Hi,
I've recently noticed this paper as well and I am quite curious to check it.
Anyway did you manage to clone your fragment using the protocol attached by Jorge?
Any chance you gave another try to the FastCloning technique?
Thanks
FastCloning did not work out for me, however, primer extension PCR cloning (method posted by Jorge) worked like a charm.
In order for this to work properly by the published method you need to transform into a RecA+ strain to ensure recombination. DH5a and other such strains are RecA1 (function blocking point mutation in the gene) which will mean the efficiency is low to nothing. I know that the original paper states they used RecA1 strains but this must be a mistake. It does state in the paper that there is some 'unknown' mechanism by DpnI action, again I think it is highly unlikely. I have used this before with about an 80% success rate with only very occasion errors if I use a high fidelity polymerase like Pfu.
I had the same problem to set the FastCloning up in the lab, so I think I can give you a piece of advice regarding the technique:
- Li et al. 2011, obtained all their results by amplifying with 17 bp homologous flanking regions (HFR), they transformed in chemically competent XL-10 Gold E. coli (which is a recA negative strain). http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-11-92
- Jacobus and Gross, 2015, obtained the same results amplifying with primers containg from 20 to 30bp HFR, and transformed in a DH5alpha strain (recA1). http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119221
According to Xia et al. 2014, even the Quickchange method is probably an homologous recombination based event. https://nar.oxfordjournals.org/content/early/2014/11/15/nar.gku1189.full.pdf
In my experience, when you use 30 bp or longer HFR, supercompetent cells are not needed; conversely, when HFR are less than 20 bp length you will need to use supercompetent cells. And last, but not least, the process seems to be independent of the number of thermal cycles.
There is perhaps a no-yet described recombination system in E coli.
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