I would like to create libraries with the following protocol but the second adapter ligations seems not to work:
1. MseI digestion of genomic DNA
2. Custom adapter ligation with MseI overhang on one side and A-tail on the other end
3. Ligation of Illumina Y-adapters to sequence the library on the MiSeq (after step 9 in the attached figure)
While the first ligation appears to work, the second ligation does not at all. I tried then to End-repair and A-tail the fragments (with the first custom adapter attached) but when I run PCR with Illumina primers after that, there is still no PCR product of the fragments.
What could here go wrong? T custom adapters have phosphorylated 5'-ends and A-tails at the 3-end that is bound to the next base with a phosphorothioate bond (small s in the attached drawing) in order to prevent exonucleases from cutting off the A-tail.
I appreciate any kind of help.
1. MseI digestion of genomic DNA
2. Custom adapter ligation with MseI overhang on one side and A-tail on the other end
3. Ligation of Illumina Y-adapters to sequence the library on the MiSeq (after step 9 in the attached figure)
While the first ligation appears to work, the second ligation does not at all. I tried then to End-repair and A-tail the fragments (with the first custom adapter attached) but when I run PCR with Illumina primers after that, there is still no PCR product of the fragments.
What could here go wrong? T custom adapters have phosphorylated 5'-ends and A-tails at the 3-end that is bound to the next base with a phosphorothioate bond (small s in the attached drawing) in order to prevent exonucleases from cutting off the A-tail.
I appreciate any kind of help.
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