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I am working on developing a protocol for digital gene expression that will produced longer tags ranging from 15 to 250 bp (plus bp from adapters). I was wondering how important the use of a Novex 6% TBE PAGE gel is, or if anyone had tried the Illumina RNA protocols using an agarose gel instead (and if they did, what %, voltage, etc.).
All these sequences are double stranded DNA, so why does Illumina recommend so many different types of gels for different sample preps? Small RNA, DpnII, and NlaII sample preps use the 6% Novex TBE PAGE. Genomic DNA, ChiP, and mRNA-seq sample preps use 2% low-range ultra agarose. Since my fragments will be a range of sizes a bit larger than the tags generated by Illumina's protocol, I am thinking it would be appropriate to try running the library on 2% agarose for the first try. Any thought? Suggestions on what bp range I should cut out, or if I should cut out a couple bp ranges and run them as different samples for the first try would be great as well. Thanks for all the great info here!
All these sequences are double stranded DNA, so why does Illumina recommend so many different types of gels for different sample preps? Small RNA, DpnII, and NlaII sample preps use the 6% Novex TBE PAGE. Genomic DNA, ChiP, and mRNA-seq sample preps use 2% low-range ultra agarose. Since my fragments will be a range of sizes a bit larger than the tags generated by Illumina's protocol, I am thinking it would be appropriate to try running the library on 2% agarose for the first try. Any thought? Suggestions on what bp range I should cut out, or if I should cut out a couple bp ranges and run them as different samples for the first try would be great as well. Thanks for all the great info here!