Hi everyone,
I need to perform a site-direct mutagenesis on a little domain and I would like to use one of the kit available on the market, but I don't know which one.
Can someone with more experience with this technique please help me?
DEar Roberta Cittadino
when i was PHD, i used extensivelly the Quick change site directed mutagenesis of Stratagene
https://www.agilent.com/en/product/mutagenesis-cloning/mutagenesis-kits/site-directed-mutagenesis-kits/quikchange-ii-233117
it works quite well for single point site directed mutagenesis.
In thje last 10 years, in Novartsi i learned the PIPE cloning approach which can be used also for mutagenesis and which do not require any specific kit but you needed just to desing correclty the primers to perfrom a vector PCR with an high fidelisity polimerase (that do not add poly AA sequences and preferabiliy with the 5'-3' endonuclease activity) and perform the trastofmation in thermo MACH1 cells (those as the only commercial cells that in my expereince works with PIPE cloning) since the HK100 reported in the papers are not commercialli avaialbe
You can found more informations about PIPE clonnig in the attached papers and at the following links on my blog:
https://proteocool.blogspot.com/search?updated-max=2021-09-30T13:21:00-07:00&max-results=10
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
good luck
Manuele
By little domain, do you mean a small gene cloned into a vector? If so, NEB's site-directed mutagenesis kit is perfectly fine for introducing mutations using PCR. Alternatively you could perform the same reaction by performing an inverse PCR followed by ligation of a gel purified product (or remove parental DNA by DpnI digest).
I work with nanobodies and have found that the best approach has been to simply order the mutated gene fragment (~450bp) for DNA synthesis. It usually takes about a week lead time to receive (at least from Twist Biosciences), the cost is very low (~35USD for that size), and it saves time (no need to confirm the mutation, just clone it and go).
Good luck! :)
Hi,
Agilent's QuikChange II Site Directed Mutagenesis Kit (https://www.agilent.com/en/product/mutagenesis-cloning/mutagenesis-kits/site-directed-mutagenesis-kits/quikchange-ii-233117) works quite well for me. It allows for point mutations (even in several sites at once), insertions and deletions depending on the primers designed (a dedicated tool for primer designing is available online).
Using primers with mismatch bases, DMSO, high-fidelity polymerase (e.g. Q5) and DpnI enzyme is also an option if commercial kits are unavailable for you (the basis of mutation incorporation is similar to this of the kit mentioned above). However, this method might require more adjustments of PCR reaction conditions.
Good luck!
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