cloning of small double strande DNA - I NEED HELP (Jan/20/2010 )
I need help: I 'm really desperate.
I'm trying to clone the hairpin for sirna (tot 53 bp: sense, loop and antisense): I have the two strand of DNA complementary plus and final A ( on both strand ) to clone in TOPO-A plasmid (the invitrogen classical one): I do the annealing, ligation in topo A, but I don't get any positive colony at all. .. I dont' know why: I thought is was muche easier.
I need suggestions
You might need to phosphorylate the primers, either after purchase, or by ordering 5' phosphorylated primers from your supplier.
phage434 on Jan 20 2010, 08:24 PM said:
thank you phage434 for your reply.
the point is : the topoA to work needs no phosphate group at the 5' position ( the topoisomerase doesn't work with the 5' P): the datasheet is very clear about that ..
We cloned a lot of oligos into restriction enzyme digested plasmid with 100% success rate, but have not done this with Topo-A vector. We don't order phosphorylated oligos. What kind of controls you have included - controls for ligation, transformation, etc. There are many posts here discussing oligo cloning, you can just search for "oligo cloning". Here are my suggestions:
1. order your oligos from a reliable vendor (desalted is good enough, make sure they are full length)
2. After annealing, run an agarose gel along with single stranded to check if the annealing is successful.
3. Try different ligation conditions: @RT for 1 hrs or 4C o/n
4. Include proper controls in every step.
Hope that helps.