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Hello everyone, has anyone tried to modify the amount of tranposome during tagmantation using Nextera? We have already modified the protocol with good results concerning tagmentation time, but we are now considering of decreasing TD and I am worried if this would work....Does anyone know if the Rd1 and Rd2 seq primers are in the TD tube or in the TDE tube (which we do not plan to decrease)?
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I do not know what your aim is to modify the Illumina protocol. TD is 2x tagmentation buffer and if you are planning to modify its amount you will need to proportionally modify your reaction volume. TDE tube contains transposon complex which inserts 19 bp transposase recognition sequences appended with 14-15 nt overhangs to DNA in tagmentation reaction that are used during PCR to add sequencing and flow cell binding motives and index sequences. R1 and R2 sequencing primers come with sequencing kit (MiSeq) or as separate kit which are added to SBS reagents in HiSeq systems. -
I am sorry I meant decreasing TDE!!!!!!!!!!!!!!!!Comment
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Decreasing TDE without any other modification will results in larger fragments. If you reduce TDE only , then you will not have enough buffer to use it in other reactions. So, if you are aiming for larger fragments it would make more sense to reduce reaction volume (TD, TDE and input DNA) and keep the input DNA amount the same.Comment
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yes I am aiming for larger fragments! thanks for the info!Comment
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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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