Get the Most out of Your µDrop Plate
Contents
1. Plate type of a measurement device the theoretical detection limit is always determined by the instrument i e the precision of the blank LOD For example with Multiskan GO which has a precision specification of 0 003 Abs the theoretical detection limit is 3 0 003 50 pg ml 10 mm 0 5 mm 9 pg ml b Linear range Because of the short pathlength the concentration that can be measured with the pDrop Plate without dilutions is tens of times higher than with a normal spectrophotometer For example Multiskan GO can linearly measure from a cuvette up to 2 5 Abs which in case of dsDNA is theoretically 2 5 50 pg ml 125 pg ml With the pDrop Plate the measurable maximum concentration depends on the photometer e Multiskan GO 2 5 50 pg ml 10 mm 0 5 mm 2500 pg ml e Varioskan Flash 4 50 pg ml 10 mm 0 5 mm 4000 pg ml c Precision Precision means the repeatability of successive measurements In photometric measurements it is normally given as standard deviation SD In pDrop plate measurements it depends on the photometer s precision specification Because of the short pathlength the absorbances caused by the low concentration samples are really low only a few milli absorbances This means that the variation SD of these samples is naturally a lot higher than for the same samples on a longer pathlength How to avoid low precision Because of the small measurement area the pDrop plate is vulnerab2. 49 6184 90 6940 or 33 2 2803 2000 Thermo SCIENTIFIC Part of Thermo Fisher Scientific
3. 6 well plate the measured value differs less than 2 from the linear value at 450 nm Material The pDrop Plate is made of aluminium and quartz glass similar to microscope glasses The low volume area is partly covered with Teflon PTFE Measurement The pDrop Plate does not limit the use of measurement steps in SkanIt Software compared with a normal 96 microplate The only limitation is the sample area columns 2 and 3 The USB memory stick provided with the pDrop Plate contains some ready made sessions It is also possible to perform measurements with the internal software of Multiskan GO In this case the user needs to perform possible calculations in a separate software such as Microsoft Excel Other methods Compared to the fluorometric methods the photometric method is cheaper and simpler but the major disadvantages of the absorbance method are the large relative contribution of nucleotides and the interference caused by contaminants For example it is possible to measure the nucleic acid concentration also by using fluorometric stains The fluorometric method is more sensitive e g the Quant iIT PicoGreen dsDNA reagent enables quantification down to 25 pg mL of dsDNA However it requires more assay steps and is more expensive Pathlength The pathlength of the pDrop Plate is the distance between the quartz glass surfaces i e the length of the light beam in the liquid sample Absorbance always depends on t
4. Thermo Scientific uDrop Plate Marika Raitio Thermo Fisher Scientific Vantaa Finland uDrop Plate Get the Most out of Your Drop Plate This paper aims to help you to get the best out of the Thermo Scientific uDrop Plate The Drop Plate is intended for photometric low volume and cuvette measurements The plate is compatible with Thermo Scientific Multiskan GO and Varioskan Flash instruments This document concentrates mainly on nucleic acid measurements Accuracy Accuracy of a reader means its ability to measure the true value The accuracy of the result gained with pDrop Plate always depends on the specification of the instrument it is used with Blanking Any photometric measurement device cuvette microplate or pDrop Plate always has some background absorption Therefore blank subtraction is always necessary in photometry when quantification of the sample is performed No blanking is not an option How to perform blanking There are several ways to perform blanking with the pDrop plate e Average The average of blank samples is subtracted from all other samples This can be performed within the same plate or at a separate measurement e Sample specific The blank is measured before the sample in the exactly same sample well 320 nm subtraction This is actually background correction and not blanking 320 nm is a wavelength at which the absorbance of both proteins and nucleic acids is minimal and an elevation in
5. elow 0 050 Abs If the absorbance level is too high clean the plate thoroughly The blank sample spectrum is also an excellent tool for troubleshooting and cleanliness verification Avoid touching the glass surface and always use disposable gloves when cleaning the plate Detection range For the pDrop Plate the lower part of the detection range is determined by the precision of the blank Limit of detection LOD sensitivity and the upper part by the linear range of the instrument A theoretical comparison of a dsDNA measurement with a 10 mm cuvette microplate and the pDrop Plate low volume area with an instrument which has the specified precision of 0 003 and which is linear up to 2 5 abs is shown in Figure 1 Abs 10 rem ev verte B udrog ciate low volume ares wae UV vicrociste with 5 mm osthienght 01i 1 10 100 1000 AONA concentration ug al 10000 Figure 1 The theoretical difference between the detection ranges of a cuvette 384 well microplate and the Drop Plate a Sensitivity The sensitivity of the assay is determined according to IUPAC Limit of Detection LOD LOD is the lowest amount of analyte that can be separated from the background It is calculated based on calibration curve slope vs blank 3 SD of the blank LOD means that this amount of analyte can be detected with statistical significance but not necessarily quantified as an exact value What to expect With a pDrop
6. he pathlength and when pathlength is shorter it is possible to measure higher concentrations It is therefore possible to measure higher concentrations with the pDrop Plate than with for example a 10 mm cuvette Ratios 260 280 and 260 230 Both the A260 A280 and 260 230 ratios give an indication of the sample purity Proteins especially due to Tyrosine and Tryptophan strongly absorb at 280 nm Therefore Abs280 is generally used as an indicator of protein contamination thermoscientific com platereaders Phenol and guanidium salts strongly absorb at 230nm Therefore this wavelength can be an indication of contamination of these compounds Nucleic acid samples with 260 280 ratios of 1 8 2 2 and 260 230 ratios of 1 8 2 2 are generally considered pure Magnetic beads may cause scattering and therefore 320 nm subtraction is especially recommended with samples purified with those also for the ratio calculations Replicates The use of several replicates is always recommended In general a greater number of replicates provides higher precision and thus more reliable results Sample Use only properly purified samples The 260 280 nm and 260 230 nm ratios described above are useful tools for evaluating the quality of the sample Close the lid gently to avoid splashing Bubbles or dirt on the sample may affect the measurements If the results seem erroneous 1 Check that there are no air bubbles in the sample 2 Check that a
7. le to bubbles and extra particles To ensure high precision e Mix the samples thoroughly e Clean the plate thoroughly e Preferably use the reverse pipetting technique What to expect on Drop plate Five dsDNA samples were measured with a semimicro cuvette 384 well UV plate and pDrop Plate Table 1 UV semimicro Cuvette uDrop Plate 384 well UV plate ug ml ug ml CV ug ml CV 1 1 3 0 9 below range 50 0 9 10 2 5 1 4 8 10 4 3 1 3 23 2 23 5 5 22 6 0 5 4 114 8 118 5 1 113 2 0 5 5 Over range 588 1 1 Over range Table 1 Five samples measured with a semimicro cuvette 384 well UV plate and Drop Plate For the plates n 8 Extinction factor The 384 well plate volume was 50 pl and the pathlenght therefore The extinction factor is needed to calculate the 5mm Over range means that the absorbance value is outside the photometer s linear range Below range means below the theoretical sensitivity limit Empty plate Do not measure empty wells as the reflections may cause strange results The reflection is a lot stronger between quartz and air than between quartz and water This is even more important with the pDrop Plate compared with a normal microplate due to a doubling of the reflecting surfaces Evaporation Because of the very small sample volume evaporation plays a more significant role than in for example microplate measurements What to expect Evaporation will result in highe
8. ll the quartz surfaces are clean and unscratched Session A session refers to a measurement protocol and possible corresponding data in SkanIt Software Spectrum It is not necessary to measure the spectra of all the samples but it is a very good troubleshooting tool Volume The usable volume of a sample for the pDrop plate is 2 10 pl The greater the volume the smaller the effect of evaporation 2012 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details North America USA Canada 1 603 595 0505 USA toll free 800 345 0206 Italy 39 02 95059 552 Netherlands 31 76 571 4440 Europe Nordic Baltic countries 358 9 329 100 Austria 43 1 801 400 Russia CIS 7 495 739 76 41 Belgium 32 53 73 42 41 Spain Portugal 34 93 223 09 18 Finland 358 9 3291 0200 Switzerland 41 44 454 12 12 France 33 2 2803 2000 UK Ireland 44 870 609 9203 Germany national toll free 08001 536 376 TTMluDrop_0512 Germany international 49 6184 90 6940 Asia Australia 613 9757 4474 China 86 21 6865 4588 or 86 10 8419 3588 China toll free 800 810 5118 400 650 5118 India 91 22 6716 2200 Japan 81 3 5826 1616 Korea 82 11 796 7771 Other Asian countries 65 6872 9717 Countries not listed
9. r concentrations For example very slowly pipetting eight sample replicates would cause the first replicates to give remarkably higher results than the last replicates How to avoid evaporation e Pipette as rapidly as possible e Measure the plate immediately after pipetting e Use an eight channel pipette whenever possible e Though 2 pl is adequate to fill the sample area use a slightly greater volume whenever possible e Do not perform long kinetic measurements concentration of a sample according to the Lambert Beer equation For example an Absorbance of 1 0 at 260 nm correlates to 50 pg ml of dsDNA The following table gives the average values generally used for nucleic acids Extinction 1 0 Abs Nucleic acid coefficient corresponds to ug ml cm 1 ug ml dsDNA 0 020 50 ssDNA 0 030 33 RNA 0 025 40 Oligos 0 033 30 Example a blank subtracted abs value of 0 045 of a dsDNA sample on a pDrop Plate with pathlength of 0 51 mm would mean 0 045 50 pg ml 10 0 51 44 1 pg ml Linearity Linearity describes the difference between the instrument s measurement values and a known standard over the full range of the expected values For photometers the upper part of the absorbance area high concentrations is especially difficult because less and less photons reach the detector For example the following specification 0 2 5 Abs 96 well plates at 450 nm 2 means that up to 2 5 abs on a 9
10. the 320 nm value indicates turbidity in the sample This subtraction is recommended especially when magnetic beads have been used for the nucleic acid purification since any beads remaining in the sample strongly scatter light What to expect Sample specific blanking gives better performance if the sample area is somehow affected e g scratched or not properly cleaned In normal conditions average blank is probably the most convenient Carry over In the case of a pDrop Plate carry over means that the plate is not properly cleaned and some leftover from the previous sample affects the following measurement How to avoid carry over Clean the plate thoroughly after every measurement as instructed in User manual or below in this document Cleaning The pDrop Plate can be simply wiped clean between the samples in a series Wipe the samples with a dry lens tissue or soft cloth The lens tissue must not contain silicon Between or in the measurement series the low volume area should be cleaned with a lens tissue dampened with de ionized distilled water and after that with 70 ethanol The cleanliness of the plate can be verified with a buffer measurement e Pipette buffer into each well Check that there are no air bubbles in the sample Use the buffer and measurement wavelength used in the daily routine measurement e Measure the absorbance in each well e The absorbance value of a clean sample position should be low b
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