Hi guys,
I have recently been performing runs on the Roche GS Junior platform. I am using the platform for viral discovery from affected samples and have performed a few runs so far.
To date my results have varied depending on the run. I initially ran into a problem during the quantification step. I use the Quantus Quanitfluor fluorometer which requires a minimum volume of sample + dye of 200uL to obtain an accurate reading. Therefore, I cannot use the RL standard protocol detailed in the Rapid Library Preparation manual as its final volume is 50uL. Any attempt to scale this up would be inefficient cost wise.
Communicating with a Roche representative I was advised to use PicoGreen for my quantification along with a customised spreadsheet from Roche to work out my dilutions to obtain 2 x 10E6 molecules/uL.
My problem is that quantifying my samples this way seems to lead to an underestimation of DNA for the emPCR stage. This is evident as I have carried out the emPCR stage using 10uL of library quantified this way and the enrichment step produced a very small amount of enriched beads - barely visible in the tube.
I was adivsed to increase the amount of molecules per bead in order to obtain an appropriate amplification which I will be carrying out tomorrow.
I would like to know if there are other viable alternatives to the RL standard to obtain a more precise quantification, or if I could somehow modify the protocol for the RL standard to suit my needs and still be cost-efficient.
Apologies if any of this seems like a silly question.
Thank you!
I have recently been performing runs on the Roche GS Junior platform. I am using the platform for viral discovery from affected samples and have performed a few runs so far.
To date my results have varied depending on the run. I initially ran into a problem during the quantification step. I use the Quantus Quanitfluor fluorometer which requires a minimum volume of sample + dye of 200uL to obtain an accurate reading. Therefore, I cannot use the RL standard protocol detailed in the Rapid Library Preparation manual as its final volume is 50uL. Any attempt to scale this up would be inefficient cost wise.
Communicating with a Roche representative I was advised to use PicoGreen for my quantification along with a customised spreadsheet from Roche to work out my dilutions to obtain 2 x 10E6 molecules/uL.
My problem is that quantifying my samples this way seems to lead to an underestimation of DNA for the emPCR stage. This is evident as I have carried out the emPCR stage using 10uL of library quantified this way and the enrichment step produced a very small amount of enriched beads - barely visible in the tube.
I was adivsed to increase the amount of molecules per bead in order to obtain an appropriate amplification which I will be carrying out tomorrow.
I would like to know if there are other viable alternatives to the RL standard to obtain a more precise quantification, or if I could somehow modify the protocol for the RL standard to suit my needs and still be cost-efficient.
Apologies if any of this seems like a silly question.
Thank you!
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