Insert 2 single strand DNA into a vector - (Jun/20/2006 )

hi friends


i need help i have insert 2 single strand DNA(separately)

each one 90bp i want to clone it to vector 6 kb

but i got each of them separately

How can i make them double strand is there any protocol to do it ?


thank you

hi
if vector is double strand (which i assume) and if you have cloned your "single strand" in this vector, that's a double strand !
may you explain little more about your exp? (vector at least...)

if you check this link you may find a protocol that will work for your needs. there are kits available, as well as a handful of published homebrew methods

I think you need to generate double-stranded DNA before you can clone it. I also think you might want to consider adding restriction sites to your DNA, unless you wish to blunt the ends and clone it that way...there are many options

good luck

my vectro is double strand (gwiz-blank) and i did not clone my "single strand" in this vector

i want to make the (2)single strand a double strand to ligate them in the vector

ok
so procedure for that is annealing of oligos...
see this thread : http://www.protocol-online.org/forums/inde...880&#entry11880

thank you very very very very very very very very very much

fred_33 and aimikins

I do it jus like fred (STE buffer, 95° x 5 ', then rp)

With this protocol, the efficiency or alignment about 50 %.

For ligation I use 50 or 100 ng of vector and a lot of insert (alignment reaccion), around 0.5 or 1 ug . It work very good for me.