5' adaptor ligation fail - (Jul/22/2007 )

Dear friends,

I have recently try to clone miRNA from the cells following Bartel's protocol. I got success in 3'adaptor ligation, but totally failed in 5' adaptor ligation. I see nothing on the the 12% PAGE even the RNA control (instead of you radioisoptope labeling internal control, I use the RNA oligo (18-24 nts) to do the experiment in parallel as a control to cut band). Is any one have suggestion how to improve the efficiency on 5' adaptor ligation. I also try the "smart cloning protocol" from clontech. seem to have band but no microRNA trapped inside.......
Do you have any idea.....
by the way, do we see the bands (no isotope) on the PAGE after 3' or 5' adaptor ligation?
I try to do the similar experiment for more than 5 times but also failed.
Hope you can save my life. Thank you very much.....

Do you use RNA oligo or oligo with RNA residue at 3' part for such ligation?
By the way, Smart technque need specilized adapter for cDNA extension, therefore, you'd better used adapter oligos from Smart kit...

Dear rye,

I use the hybrid one as 5'adaptor. That mean 5'DNA/RNA3'. I am still don't know the reason.

Did you try Smart kit for miRNA cloning before? How is the performance..... I am still doubt that the extra CCC from the RT reaction is enough for annealing and cDNA extension....

You might cut gel around the range of expected ligation size, then RT and PCR to see whether you can amplify small RNA cDNA.
as to smart oligo, I was told it is specially modified or with specific modification/chemistry. I am sure it is ture, but I did fail in similar work using home_made smart adapter.
By the way, is your control RNA oligo with 5' P?