Hello everyone
We've been struggling with our project due to low efficiency rates + check the insert is OK and not in reverse + PolTaq did not introduce a new mutation... and it has come to our attention that we could try this ligase-free cloning methog called in-fusion cloning, in which you work with complementary ends of both the insert and the plasmid, apparently. We wouldn't like to waste our time trying new stuffs out of the blue, so maybe anyone has a feedback for us. We would be very grateful.
Thanks again
Dear Beatriz
I'm currently using the PIPE cloning, which was developed by NIBR (Novartis) which is an enzime free cloning method very similar to the In-fusion.
(I suspect that IN-fusion is just a commercial version of the PIPE)
In my hand it work very well to insert up 3Kb (i have limited experience with larger insert) and for delection and single point mutagenesis.
The only limit of this method is that if you cannot use the HK100 strain (which is tihnk is Novartis proprietary) the only alternatrive commercial E.coli strain that i found able to perform it are the MACH1 ot thermo (chemically competent).
Attached you can find the articles of the scientist that developed this method.
If you are interested to know more detail about it and primer desing you can find 2 video on my blog ProteoCool (https://proteocool.blogspot.com/) at PAGE1.
good luck
Manuele
Yes we use infusion cloning quite regularly in our lab and it is working good so far. Just follow the company's cloning protocol and the cloning should work nicely!
Hope this helps
I haven't had luck with in-fusion unfortunately. I have used NEBuilder HiFi DNA Assembly ( https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly?redi=nebuilderhifi ) and it works wonderfully. If you've heard of Gibson Ligations, this is essentially an updated version which is less error-prone. If you don't end up having luck, check this assembly kit out!
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