Prbbbb:dna measurement on plate v1

(Difference between revisions)
 Revision as of 07:02, 20 May 2009 (view source) (→Calculation)← Previous diff Current revision (23:20, 6 January 2011) (view source) (24 intermediate revisions not shown.) Line 3: Line 3: ==Overview== ==Overview== + Rather than "nanodropping" every well by hand, we measure DNA concentrations on a plate reader. + + '''Accuracy''' + The results agree with nanodrop measurements to within 5% deviation. + + '''Limits''' + Concentrations down to 10 ng/µl can be reliably measured. The volume should be 20 µl or more per well. The samples are later recovered for dilution and storage. + Using Greiner 788876 plates, I have had good luck down to 10 ul (DTAE). ==Materials== ==Materials== Line 10: Line 18: *384 well UV transmissive plate with flat clear bottom *384 well UV transmissive plate with flat clear bottom ** we use Greiner UV star Cat # 781 801 ** we use Greiner UV star Cat # 781 801 + *Greiner 788876 are also recommended for even lower volumes (although they are kind of expensive, about $10/plate and only come in packs of 80) * miniprepped DNA, at least 30 µl * miniprepped DNA, at least 30 µl ==Calculation== ==Calculation== + + Absorption is proportional to the path length through your sample. On a microtiter plate, + this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions + of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we + tried, this works surprisingly well and there is no need for calibration curves. + You can also calculate the pathlength empirically on a well-by-well basis by getting the absorption at 977 and 900 and then using the equation. This techniques may be superior because it should partially control for pippeting error as well as sample adhering to the edges of the wells. + $d=(A977-A900)/0.18 +$ '''parameters''' '''parameters''' - * well dimension: flat bottom 3.3 mm x 3.3 mm * DNA extinction coefficient e: 50 [(ng * cm) / µl] * DNA extinction coefficient e: 50 [(ng * cm) / µl] - * path length: + * well dimension: flat bottom 3.3 mm x 3.3 mm - d = V / (3.3^2 * mm^2) + - V... volume per well in µl + * path length in mm (d') or in cm (d): + + $+ d' = V / (3.3^2 * mm^2) +$ + + $+ d = d'/ 10 +$ + + (V... volume per well in µl) '''formula''' '''formula''' - c = (A * e) / d + Once the path length d (in cm) is known, the DNA concentration is calculated from the measured absorption A + (corrected by the blank absorption A0): + + $+ c = \frac{(A-A0) \cdot e}{d} +$ * c... DNA concentration in ng / µl * c... DNA concentration in ng / µl * A... Absorbance @ 260 nm * A... Absorbance @ 260 nm + * A0...Absorbance of blank sample (same amount, same buffer/water) * d... path length in cm * d... path length in cm + * e... extinction coefficient in ng*cm/µl ==Procedure== ==Procedure== # Pipette exact volume of DNA samples into 384 well plate # Pipette exact volume of DNA samples into 384 well plate + #* multi-channel pipetting from a 96-well plate works (SABiosciences PA-384 guides help when x-fering between 96 and 384 well plates) + #* note, the pipetting is the biggest source of error # measure 260 nm and 280 nm absorbance on plate reader # measure 260 nm and 280 nm absorbance on plate reader # Pipette sample back into storage plate # Pipette sample back into storage plate - # calculate DNA concentration from formula above (compare to reference) + # calculate DNA concentration from formula above - + ==Notes== ==Notes== Line 43: Line 77: raik: raik: - This protocol is quick and robust in my hands. + We are just starting to use this protocol -- share your experience! Current revision Contents Overview Rather than "nanodropping" every well by hand, we measure DNA concentrations on a plate reader. Accuracy The results agree with nanodrop measurements to within 5% deviation. Limits Concentrations down to 10 ng/µl can be reliably measured. The volume should be 20 µl or more per well. The samples are later recovered for dilution and storage. Using Greiner 788876 plates, I have had good luck down to 10 ul (DTAE). Materials • Tecan plate reader • (or any reader that accepts 384 plates and can measure at 260 and 280 nm) • 384 well UV transmissive plate with flat clear bottom • we use Greiner UV star Cat # 781 801 • Greiner 788876 are also recommended for even lower volumes (although they are kind of expensive, about$10/plate and only come in packs of 80)
• miniprepped DNA, at least 30 µl

Calculation

Absorption is proportional to the path length through your sample. On a microtiter plate, this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we tried, this works surprisingly well and there is no need for calibration curves. You can also calculate the pathlength empirically on a well-by-well basis by getting the absorption at 977 and 900 and then using the equation. This techniques may be superior because it should partially control for pippeting error as well as sample adhering to the edges of the wells. d = (A977 − A900) / 0.18

parameters

• DNA extinction coefficient e: 50 [(ng * cm) / µl]
• well dimension: flat bottom 3.3 mm x 3.3 mm
• path length in mm (d') or in cm (d):
d' = V / (3.32 * mm2)
d = d' / 10

(V... volume per well in µl)

formula

Once the path length d (in cm) is known, the DNA concentration is calculated from the measured absorption A (corrected by the blank absorption A0):

$c = \frac{(A-A0) \cdot e}{d}$
• c... DNA concentration in ng / µl
• A... Absorbance @ 260 nm
• A0...Absorbance of blank sample (same amount, same buffer/water)
• d... path length in cm
• e... extinction coefficient in ng*cm/µl

Procedure

1. Pipette exact volume of DNA samples into 384 well plate
• multi-channel pipetting from a 96-well plate works (SABiosciences PA-384 guides help when x-fering between 96 and 384 well plates)
• note, the pipetting is the biggest source of error
2. measure 260 nm and 280 nm absorbance on plate reader
3. Pipette sample back into storage plate
4. calculate DNA concentration from formula above

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: We are just starting to use this protocol -- share your experience!