Has anyone had success generating Adaptor1-TAG-Adaptor2 products for Solexa Sequencing?
I am trying to get this working and am only able to make Adaptor 2 concatamers. Basically, I extract RNA from tissue with trizol. Bind the rna to polyT dynabeads. Perform first and second strand synthesis. Cut with NlaIII. Ligate Adaptor 1. Cut with MmeI. Ligate adaptor 2. Perform PCR with a primer pair that binds to sites in adaptor 1 and adaptor2, respectively.
I consistently a ladder starting at 80bp and increasing in ~40bp increments (eg, 85bp, 120bp, 155bp, 185bp, 225bp, 265bp, 300bp, etc).
Curious to see if anyone else has experienced this and if so, what they did to troubleshoot or get the adaptor2 to ligate to the Adaptor1-Tag oligo.
Thank you.
I am trying to get this working and am only able to make Adaptor 2 concatamers. Basically, I extract RNA from tissue with trizol. Bind the rna to polyT dynabeads. Perform first and second strand synthesis. Cut with NlaIII. Ligate Adaptor 1. Cut with MmeI. Ligate adaptor 2. Perform PCR with a primer pair that binds to sites in adaptor 1 and adaptor2, respectively.
I consistently a ladder starting at 80bp and increasing in ~40bp increments (eg, 85bp, 120bp, 155bp, 185bp, 225bp, 265bp, 300bp, etc).
Curious to see if anyone else has experienced this and if so, what they did to troubleshoot or get the adaptor2 to ligate to the Adaptor1-Tag oligo.
Thank you.
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