8, Measure fluorescence of sample using instrument parametersthat correspond those ‘sed when generating standard curve (see steps 206). To minimize photobleaching effects, keep time fr fluorescence measurement constant fra amples 9, Subwractfuoresence valve of reagent bank from that of each sample. Determine DNA concentration ofthe sample fom standard curve 10, I desired, repeat assay using a diferent dilution ofthe sample oeonfiem results. QUANTITATION OF NUCLEIC ACIDS USING A MICROVOLUME SPECTROPHOTOMETER WITHOUT THE USE OF CUVETTES OR ‘CAPILLARIES. ‘The NanoDrop ND-1000 spectrophotometer uses a patented sample retention system (Fig, A3D2) that holds 1 ul of sample without tbe need for wadtional containment devices such as cuvetes and capillaries. Using ler opie technology and surface tension, the sample is ed in place Between two optical surfaces that define the pathlength in 1 vertical orientation, Removal of xed containment devices from the system allows the pathlength to change in realtime fora given sample. During each measurement cycle, the sample js assessed a both a I-m and 0.2-mm path, providing an extensive ‘dynamic range (2 ng/jl 10 3700 ngliul for dsDNA; Table A.3D.5). This essentially ‘liminales the need to perform dilations oro make any assumptions regarding the sample txoncentation prior to the measurement Diet coupling ofthe sample to the opies of the spectrophotometer removes interference caused by incident light and trnsmited light passing through containment walls of traditional cuvetes, mirocell were, and capillaries. Preparation forthe next sample only requires wiping ofboth optical surlaces ‘with a common laboratory wipe, The time nceded for performing ditions and for cleaning the cavette and microcell devices, as well as the possibility of damaging sich devices, is eliminated, Total measurement cyele time, including preparation and removal ‘ofthe sample, is ~¥0 sec. The ease of use ofthis protocol not only makes ita feasible ‘option formal volume analysis, but alsoa practical allematve forall spectrophotometic Matriais [Nucleic acid sample to be quantitated ‘Water or buffer in which sample is dissolved 'NanoDrop ND-1000 Spectrophotometer(NanoDrop Technologies, Ine Inpinnsnanodrop.com) Clean the upper and lower optical surfaces ofthe microspectrophorometer sample ‘tention system a follows Ppet | to 2 yl of clean deionized water onto the lover ‘optical surface, Close the lever arm ad tap ita few times to bate the upper optical surface. Lift the lever arm and wipe off both opial surfaces with a Kimvipe. Wong he pedestals between meatrements it wucly ficient to prevent sample car. over and avoid residue bul A final leaning ofl surfaces wth deleted water It recommended afer each users ast meatrenent. After large manber of samples, theareas around he pedestals shoul be cleaned thoroughly preven previous sample {ram being wiped back onto the pedestal, ecting lnlevel measurements. I may be “advisable clean the measuremen surfaces with ae (a above afer pariclarly Inghcoacentaion somes The NanoDrop ND-1000specrophatonter i compaile with mos solvents typically sed in Bf cence laborers, eluding methane ethanol npropan isopropanol, butanol, acetone ether, chlrafrm, carbon terachiride, DMSO, DME. acetone THE, tone, hetane, benzene, odin hydride, sdionhypoeMorite (leach) ane Ct ae HNO aed dre cea ALTERNATE ‘prorocoL.s ‘ecm ABD9loading piste - z a btsample ty sample ip ce measuring ‘enon fan ame Cab astator Figure A302 The NanoDropND-1000 Spactophelamear micivaune sample eteiion system (A) sare valu {of lls spensed ono ho ower opical suo (B) Oreo toinsturantiover ars lowered, he upp pte race “angags mh he sample, forming iq coun wt pth ang tins bythe gap between to wo opal erage ‘Bung aacn maasuremen, ho spies assoeead at boa -mm and O2-rm path, proidng abe dar range of ‘ce ae dtocton, CAUTION: Hydofuoric aid (HF) mst not be wet clean the NawaDrop inset asthe florid ion wil dsl he quarter ote cable. (Open the NanoDrop software and select the nuclei acids module Initialize the spetrophotometer by placing Il clean water onto the lower optic surface, lowering the lever arm, and selecting “initlize” inthe NanoDrop sofware, (Once initialization scomplete~10 sec, clean both optical surfaces witha Kimipe Perform a blank measurement by loading 1 ul deionized water or buffer and seleting “blank” Once the blank is complete, clean bath optical surfaces with a Kimvipe, [As on a tradtionalspectophormeer, the Hank wll be subiracted from subsequent Imeasiements. Todeermine the contain ofa blank such ara spice mean the uf agabst dlontzed water asthe Blank. Ifthe baer der not comb to the ‘Aso, ten delnied water will supe asthe blank. The waer or buffer should ays bemeatared env hat thensramen hasbeen ered properly The measrement of ‘he water or bufer should be er or very ease 0290 All measuremests are owomaicalynormaied 340 nn. Measure the nucleic acid sample by loading 1 and selecting "measure." Once the ‘measurement is complet, clean bath optical surfaces with Kime, The measurement ond subsequent concemaion collation volume independent. The sample ony neds to bridge te gp bereen he vo opie surface for a measrenent ovbe made Plld experience indeares tha It Yolune is sefiien 0 ence Fepo= tei of aqueous soliton of mci acas, Volumes of? may be wed fr samples ‘spec of omc substances tha ey fet surface tenon. -Ensare sat the appropriate constant x been chosen for he mesarement (50 fr {DNA or 40 for RNA}. The sofware entomarcaly afelates the mille wl com ‘enpation fhe caleltion ix being done by han the Ay represented as a path for comenence even hoo Is and 0.2-mm pasa cals se dng the easement ele) caret sss Mak Bey