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Old 12-15-2014, 05:53 AM   #1
Location: UK

Join Date: Dec 2014
Posts: 37
Default Quantification Alternative

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!
GSviral is offline   Reply With Quote
Old 01-12-2015, 10:09 AM   #2
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Location: Utah

Join Date: Mar 2010
Posts: 166

The problem with quantification using PicoGreen is that it will measure all the DNA is the sample, whether or not it can function in emPCR. Variation in ligation efficiency will therefore give you inconsistent performance in emPCR. The standard quantification method reads the dye attached to the adaptors, so it gives you a better measure of functional molecules. I have a couple of suggestions for you:

1. Use qPCR. I've used a qPCR kit from KAPA that works just fine, although it's a little expensive. I imagine there are others out there, but I haven't used them. You can also make some primers matching the A and B adaptor sequences and use standard qPCR reagents, but you'll need to find something you can use as a standard, which will probably require a fair bit of optimization and troubleshooting.

2. You can amplify your library after you finish it and then quantify it using PicoGreen. I do this with just about everything now. I use leftover emPCR reagents (enzyme, amp mix) and some primers I made to amplify the libraries a few cycles. I typically use 1 ul of the library in a 25 ul reaction and amplify 5-10 cycles using the same cycling program that is used for emPCR. It's important not to let the reaction go so far that it begins to exhaust any reagents. After amplification, clean it up with AMPure beads and quantify with PicoGreen. Use your average fragment size to calculate the concentration for emPCR.
ajthomas is offline   Reply With Quote

454, pyrosequencing, quantification, rl standard

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