cloning of small double strande DNA
Posted 20 January 2010 - 08:03 PM
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
Posted 20 January 2010 - 08:24 PM
Posted 20 January 2010 - 08:52 PM
You might need to phosphorylate the primers, either after purchase, or by ordering 5' phosphorylated primers from your supplier.
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 ..
Posted 21 January 2010 - 12:16 AM
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.