Hi everyone,
I am using Illumina mRNAseq kit to prepare mRNA library for sequencing. I have two questions:
1- when you cut the band from gel after PE adaptor ligation, what range is best to cut? Illumina says "200bp" band, our core facility scientist says "200-400bp" and I personally think 190-300bp should be the best. My argument is based on the mRNA fragments produced after the fragmentation step and the length of the adapters. Fragmentation produces 60-200bp fragments (see Ambion fragmentation buffer notes) and the total length of the PE adapters is 120. This will make a range of 180-320. Is this make sense, anyone agrees with me?
2. I use the Sera-mag oligo dt particles to purify the mRNA from total RNA in two rounds of hybridization and elution. Accidentally, I made the first elution round in 1 M tris-Hcl (pH 7.4) instead of 10 mM of the same buffer. I corrected this in the 2nd elution round. I then used Nanodrop to quantify the obtained mRNA. It was around 5 ng/ul....Sound normal. Does anyone experienced this issue before. I mean should I proceed with this mRNA or not? I am going to test it with Agilent bioanlyzer, but I am still confused whether I use it or start all over again. Any advise.
I am using Illumina mRNAseq kit to prepare mRNA library for sequencing. I have two questions:
1- when you cut the band from gel after PE adaptor ligation, what range is best to cut? Illumina says "200bp" band, our core facility scientist says "200-400bp" and I personally think 190-300bp should be the best. My argument is based on the mRNA fragments produced after the fragmentation step and the length of the adapters. Fragmentation produces 60-200bp fragments (see Ambion fragmentation buffer notes) and the total length of the PE adapters is 120. This will make a range of 180-320. Is this make sense, anyone agrees with me?
2. I use the Sera-mag oligo dt particles to purify the mRNA from total RNA in two rounds of hybridization and elution. Accidentally, I made the first elution round in 1 M tris-Hcl (pH 7.4) instead of 10 mM of the same buffer. I corrected this in the 2nd elution round. I then used Nanodrop to quantify the obtained mRNA. It was around 5 ng/ul....Sound normal. Does anyone experienced this issue before. I mean should I proceed with this mRNA or not? I am going to test it with Agilent bioanlyzer, but I am still confused whether I use it or start all over again. Any advise.
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