How to insert one single nucleotide into a plasmid? - (Dec/12/2005 )

I did stupid thing, after cloning a sequence into a plasmid, just find out not in frame with the following sequence. just need insert one single nucleoacid will be ok. Any method to insert only one nucleoacid? or I need do cloning from the start

---

site directed mutagenesis should do the trick

---

Well if you obtain your sequence by pCR I would rather redo it than strat with Mutagenesis cause the process need some several experiments to be correct


Other possibility is to design two sets of primers flanking the target sequence and introduce a single base and then fix the mutation but you will still need to reclone the insert I guess !


Pesji

---

cry cry wuwuwuw
can I ask a stupid question, there are so many ATG in my sequence, why only recognize the first ATG then translate the whole sequence, why the promoter will not recognize some ATG in the middle?

---

That happens from time to time depending of the flanking amino acid, the efficency of the promotor, the type of celles you use for expression etc...


Was it such a long cloning that you're discouraged to do it again ?

Pesji

---

in general if expressed in e.coli the first ATG next to the ribosomal binding site should be were translation starts (mostly)

---

I mean in the mammalian cells. such as CMV promoter, then what is going on got so many ATG in between. what will happen

---