I am currently having some issues getting a good yield for my three-way splint ligation. I am using a DNA splint that covers the entire length of all three oligos. I get some full length RNA, but I would like to increase the yield. The 5'-end and the middle oligos ligate pretty well, but the middle and 3'-end don't seem to ligate well. The 3'-end also seems to be phosphorylated without any issues either.
Looking for possible things to check, modify, or improve upon.
This link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1624903/
Regards
You could try adding 5-10% PEG8000 and 3-10% DMSO.
This helped (5% PEG and 3% DMSO) in our case, even though we ligated an RNA to a DNA-oligo. To reduce unwanted secondary structures you might try to denature the RNAs at 95°C for about 5 minutes and then chill them quickly on ice.
You could also try different splint oligonucleotides. If the RNAs are produced by in-vitro-transcription then the 3'-end might not be homogeous. Polymerases tend to add nucleotides to the 3'-end. You can circumvent this problem by using a mixture of splint oligos that contain the complementary sequences to the 3'-end and 5'-end plus/minus a degenerated nucleotide between the two as in Moritz and Wahle 2014 (Article Simple methods for the 3 ' biotinylation of RNA
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