Hi. I think my question is a relatively simple one, but i am struggling with the calculation and would like some advice. My sample measured 2.84 ng/ul on the Qubit. The volume of that sample is 5244 ul (5.2 ml).... Large, I know! But this is a pooled sample consisting of 103 barcoded samples. From my calculations, I understand that I have 2.84 ng/ul X 5244 ul = 14893 ng = 14.8 ug (correct?). But now, I must submit to the sequencing centre, 50ul of the pool with a concentration of at least 5ng/ul. How do I do this conversion/calculation? Any help would be greatly appreciated!
2.84 ng/ul X 5244 ul = 14893 ng = 14.8 ug - correct.
Everything you need for all conversions is the formula relating amount (N), volume (V) and concentration (C):
C = N / V
(concentration is amount per volume) You solved this by N, getting N = C x V, to calculate the total amount of the solution with given C=2.84ng/ul in a volume of V=5422ul.
Your concentration (2.84 ng/ul) is smaller than the minimum concentration required by the centre (5ng/ul). So you will need to concentrate the samples (speed-vac, reduce the volume to less than 50%).
Given you want 50ul with a conc of 10ng/ul (twice the minimum required conc), this is how you can proceed:
The desired amount is N = C x V = 10ng/ul x 50ul = 500ng.
This amount is contained in V = N / C = 500ng / 2.84ng/ul = 176ul of your sample.
Take this volume of your sample and concentrated it in a speed-vac to a volumen of less than 50ul.
Measure the obtained volume of the concentrated sample (good enough to use a pipet for this purpose) and adjust the volume to 50ul by adding water or buffer.
Done.
I don't recommend to completely dry the sample and resolve it in 50ul water/buffer because: complete drying takes unneccesaryly long, it may be slightly harmful for the quality, and the resolving may not work well (when the sample got "too dry").
2.84 ng/ul X 5244 ul = 14893 ng = 14.8 ug - correct.
Everything you need for all conversions is the formula relating amount (N), volume (V) and concentration (C):
C = N / V
(concentration is amount per volume) You solved this by N, getting N = C x V, to calculate the total amount of the solution with given C=2.84ng/ul in a volume of V=5422ul.
Your concentration (2.84 ng/ul) is smaller than the minimum concentration required by the centre (5ng/ul). So you will need to concentrate the samples (speed-vac, reduce the volume to less than 50%).
Given you want 50ul with a conc of 10ng/ul (twice the minimum required conc), this is how you can proceed:
The desired amount is N = C x V = 10ng/ul x 50ul = 500ng.
This amount is contained in V = N / C = 500ng / 2.84ng/ul = 176ul of your sample.
Take this volume of your sample and concentrated it in a speed-vac to a volumen of less than 50ul.
Measure the obtained volume of the concentrated sample (good enough to use a pipet for this purpose) and adjust the volume to 50ul by adding water or buffer.
Done.
I don't recommend to completely dry the sample and resolve it in 50ul water/buffer because: complete drying takes unneccesaryly long, it may be slightly harmful for the quality, and the resolving may not work well (when the sample got "too dry").
Nice answer Jochen~! Not evaporating the sample to complete dryness is an excellent touch.
- Badges
- Science topic
- Similar topics
- Applied Mathematics
- Calculations