Problem with restriction enzyme NdeI - How many bases do I need? (Sep/11/2007 )
I'm designing a long primer with a recognition site for NdeI at 5' end. I was told that I should add extra bases next to those reognized by NdeI at the 5' end or the enzyme would probably fail to cut my PCR product. The question is which base and how many do I need do add?
Looking foward to your kind help. BOW!
P.S.
I have checked the WEB site of NEB, apparently the maker didn't mention anything about this issue inside their catalog.
Looking foward to your kind help. BOW!
P.S.
I have checked the WEB site of NEB, apparently the maker didn't mention anything about this issue inside their catalog.
The NEB website have very helpful notes for this. Here: http://www.neb.com/nebecomm/tech_reference...ized_vector.asp
and also: http://www.neb.com/nebecomm/tech_reference...nucleotides.asp
I usually put two random nucleotides at the 5' end and never had problems, but apparently (if you check the sites Almasy provided) the number and types of nucleotides that you insert can make a big difference. Learning new stuff every day 
For NdeI, a minimum of 7bp is required at each end, for the site to be cut with any efficiency. I would use 8bp. I would also use a mixture of nucleotides rather then a string of mononucleotides.
according to neb webpage "cleavage close to DNA ends, NdeI requires 8bp each side to efficiently cut.
Yeah, you need 8 bases, so I’ll prefer don’t add so many extra-bases but first clone the PCR product in a cloning vector (pTOPO, pGEMT, pJET or any) and then digest.
It seems adding an extra 'GGAATTC' to the 5'end of the primer next to the NdeI site and then digesting the PCR product overnight may be a good choice. Thaks a TON to everyone! I love this forum so much!