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We are currently trying to produce a cDNA library for sequencing from 100ng of total RNA.
From the illumina webinar on DSN normalisation, it appears that we perform all the steps in the 'Sample Preparation Guide' (except poly-A selection) and then proceed to denaturation, renaturation and DSN treatment, followed by purification with SPRI beads, PCR to enrich the fragments, and then a final purification with the beads.
Our question is this: Is it necessary to cut the 200bp band (one of the steps in the Sample Preparation Guide) when we are going to use the SPRI beads later (following DSN treatment)? The reason we are asking this is that these beads are known to be selective for a certain range of cDNA fragment lengths, so we thought that perhaps the gel-cutting is unnecessary in this case.
Any help would be greatly appreciated!
Many thanks, Adam
From the illumina webinar on DSN normalisation, it appears that we perform all the steps in the 'Sample Preparation Guide' (except poly-A selection) and then proceed to denaturation, renaturation and DSN treatment, followed by purification with SPRI beads, PCR to enrich the fragments, and then a final purification with the beads.
Our question is this: Is it necessary to cut the 200bp band (one of the steps in the Sample Preparation Guide) when we are going to use the SPRI beads later (following DSN treatment)? The reason we are asking this is that these beads are known to be selective for a certain range of cDNA fragment lengths, so we thought that perhaps the gel-cutting is unnecessary in this case.
Any help would be greatly appreciated!
Many thanks, Adam
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