I am planning to do functional analyses for a novel mutation to investigate its effect using cell based cloning. The data from literature indicate that pET+25b is the best vector to create the expression construct. My target gene is MUTYH (ENST00000450313), which spans for about 1888 bp. However, I do not know what is the most suitable restriction enzyme to do the digestion.
Thanks in advance
MYH associated polypoposis?)) Ok. Enter your sequence here http://nc2.neb.com/NEBcutter2/
If you don't find the ones, simply insert the sites that you need by PCR, with primers, that contain these sites.
If possible you should use Ndel at the 5'-end of your ORF, because NdeI site already contains ATG start codon and has the optimal space to the RBS of the plasmid! Otherwise you will add the pelB sequence to the N-terminal part of your protein. For more details you can have a closer look in the following link:
https://www.google.de/search?q=pet25b&prmd=mivn&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjZqrj-1IbSAhWTDRoKHWmtCDEQ_AUICCgC&biw=360&bih=567#imgrc=_XL7snZ91xmmpM:
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