Hi all,
I've recently come across a great review on IVA cloning here: http://www.jbc.org/content/early/2019/09/14/jbc.REV119.009109.long
Some more details here: https://blog.addgene.org/plasmids-101-simplify-cloning-with-in-vivo-assembly
I'd be really interested in giving this a go to introduce point mutations, but I was wondering if anyone else had had a go at this and could assist in primer design (which seems to be critical)?
I have an expression plasmid that encodes our protein of interest and this would be the template.
In the guide it says the 5' portion of the primer should contain homologous sequence and any modifications (i.e. the point mutation). As my point mutation would be in the middle of protein sequence I guess the 'homologous sequence' is just codons for the protein? The 3' region is also supposed to bind template, and this in my case would also just be the plasmid/protein sequence. I guess the length/Tm of the primers needs optimising, but am I missing something incredibly obvious? This seems almost too straightforward.
Thanks for any insight you can offer!
Best wishes,
James
Dear James
I think you can read about PIPE cloning (https://link.springer.com/protocol/10.1007%2F978-1-59745-196-3_6) that was developed from Novartis and on the basis of the paper that you attach i think can be classified as an IVA cloning approach.
you can found information about it and primers design on my blog:
https://proteocool.blogspot.com/
ProteoCool n°1 (Cloning methods overwiev)
and
ProteoCool n°3 (Primer Design: PIPE Cloning)
of course as you will see in the presentation it require a specific e.coli strain. To my knowledge the best strain are HK100 which are Novartis proprietary but also Mach1 seems to work quite well.
good luck
Mamuele
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