Linkers:insert ligation problem - (Jul/24/2006 )
We have some problems doing a ligation of my insert and linkers. We designed two linkers (or adaptors) in order to make compatible the ends of vector and insert.
Insert has 400 bp and Bgl II-EcoR I ends (5’-3’)
Linkers were prepared from two annelaled oligonucleotides of 12 bases, as follows:
What could be the correct ration insert:linkers?
What could be the ligation conditions? (Temp, ligase units, time…)
may be u can use some kit for ligation and get instruction from manual
Normally the ratios are 1:10 for blunt end and 1:3 for sticky ends, further conditions depend on your buffers, ligase etc. Anyway I would follow tried and tested protocol.
As hobglobin suggested I would follow tried and tested protocols.
or I would check with your oligo providers for their suggestions.
Invitrogen or IDT have these information in their websites.