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Since a long time now I have been fiddling around some Illumina Truseq primers for a custom sequencing protocol. I am using RP1 and RPI1 (and other indexing primer for my amplification from Truseq small RNA sequencing protocol). As per my plan, I am fitting the right adapters on each of the ends of the cDNA, Please refer the attached picture. I have already cross checked using adapter specific primers(exact match) however, whenever I use the Illumina TruSeq primers (RP1 and RPI1, without PS bonds) I do not see any amplification. Moreover, whenever I use RP1 and RPI1 with PS bonds I see a strong band at ~115-120 ntds. I am really tired of repeating PCRs seeing the same trend again and again. What could be the problem?
I have already tried DMSO in the reaction, lowering the temperature etc. I have also tried all the combinations (Small adapter specific primer with RP1 or RPI1) Importantly, we are planning to index the reactions.
Your suggestions and experience will be really helpful.
Biochembug
Since a long time now I have been fiddling around some Illumina Truseq primers for a custom sequencing protocol. I am using RP1 and RPI1 (and other indexing primer for my amplification from Truseq small RNA sequencing protocol). As per my plan, I am fitting the right adapters on each of the ends of the cDNA, Please refer the attached picture. I have already cross checked using adapter specific primers(exact match) however, whenever I use the Illumina TruSeq primers (RP1 and RPI1, without PS bonds) I do not see any amplification. Moreover, whenever I use RP1 and RPI1 with PS bonds I see a strong band at ~115-120 ntds. I am really tired of repeating PCRs seeing the same trend again and again. What could be the problem?
I have already tried DMSO in the reaction, lowering the temperature etc. I have also tried all the combinations (Small adapter specific primer with RP1 or RPI1) Importantly, we are planning to index the reactions.
Your suggestions and experience will be really helpful.
Biochembug
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