imjt at pop.dtu.dk
Wed Oct 20 12:31:19 EST 1999
I have made double stranded DNA from oligos 150 bases long with 20 bp
overlap. I am sure you can get longer oligos to work, but the yield
decreases when exceeding about 150 bases in length. The oligo company I use
charges extra for such long oligos because of that.
You will have to consider that the cDNA you get will be nicked once on each
strand for every other overlap (where the 5' ends anneal). You might be able
to repair that in the ligation by buying 5'-P oligos. I made my cDNA from 2
oligos only, so I did not have to worry about that.
Probably you will get quite a bit of junk after the polymerization. I
gel-purified my desired fragment before ligating in the vector to get rid of
that. Approximately 50% of my clones had the correct sequence.
NeuroSearch, Ballerup, Denmark
William Sun <william at neuro.usc.edu> wrote in message
news:7uiloq$g0a$1 at neuro.usc.edu...
> Hello all,
> Does anyone have experience assembling a cDNA from a set of overlapping
> oligonucleotides? How long can I make the primer and how big an overlap?
> Can I anneal all the primers and vector together in one step? Any advice
> or reference would be greatly appreciated.
> William Sun, Ph.D
> Metabasis Therapeutics, Inc. Phone: (619) 622-3930
> 9390 Towne Centre Drive FAX: (619) 622-5583
> San Diego, CA 92121 Email: william at alum.mit.edu
More information about the Methods