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=Quant-iT(TM) ssDNA Assay Kit= ==Materials== *Quan-iT ssDNA Assay Kit (cat. # Q32851, containing: *Quant-iT ssDNA reagent, 200x solution in DMSO *Quant-iT buffer *Oligonucleotide standard #1 (0ng/ul in TE Buffer) *Oligonucleotide standard #2 (20ng/ul in TE Buffer) *Qubit fluorometer Invitrogen Cat. Q32857 *Qubit Assay Tubes (500, Invitrogen Cat. no.Q32856) or Axygen PCR-05-C tubes (VWR, part number 10011-830)*Qubit fluorometer Invitrogen Cat. Q32857 *Sterile, distilled, DNase free water ==Starting Notes== # Make sure that all solutions are at room temperature # Do not hold the assay tubes in your hand before reading # Do not leave tubes in the fluorometer for longer than necessary as it may warm the assay solution significantly # Do not leave assay solutions exposed to light # Do not overexpose the assay solutions to more than 9 readings as solution is not photostable. # Assay solutions are not stable. Discard after 30min. Table 1. Solution {| border="1" !Standard/Sample Input !Volume (ul) of standard/sample !Volume (ul) of diluted Working Solution !Final Oligomer Conc. |- |Standards 1/2 |10 |190 |0ng/ul |- |Sample #1-6ab |4 |194 |??? |- |} Total working solution required: 2780ul Sample Information: # after 1st EtOH PPT, 1020ul # after Lamda Exonuclease, 1083ul # after QiaQuick, 420ul, diluted 10/3x # after USER & DpnII, 518ul # after size selection & Nanosep, 4047ul # after EtOH PPT, 20ul, diluted to 0.1x ==Protocol== # Set up 2 tubes for standard and 12 tubes for samples (2x each 1-6) # Label tube lids # Calculate amount of working solution required. # Load in appropriate volumes for each labeled tubes # Vortex for 5 s and spin down all tubes # Incubate for 2 minutes at room temperature # Turn on Qubit Fluorometer # Press HOME and select Quant-iT ssDNA. Press GO to initiate assay # On calibration screen, select Run New Calibration. Press GO Or select use last calibration # Insert standard#1, close lid, press GO # Remove standard#1, insert standard#2, close lid, press GO # Remove standard#2. # Repeat similarly for sample assay tubes. # Record readings given by the fluorometer. # Turn off Qubit Fluorometer after last reading: press HOME, scroll to turn off, then press GO to turn it off. ==Results== {| {{table}} border = "1" | align="center" style="background:#f0f0f0;"|'''Quant-iT ssDNA''' | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"|'''Orig''' | align="center" style="background:#f0f0f0;"|'''Orig''' | align="center" style="background:#f0f0f0;"| |- | ||Qubit Reading(ng/mL)||uL used||Dilution||Sample (ng/mL)||Sample Dilution||Sample (ng/uL)||Average (ng/ul) |- | 1a||209||4||50||10450||10||104.5|| |- | 1b||20.3||4||50||1015||100||101.5||103 |- | 2a||111||4||50||5550||10||55.5|| |- | 2b||11||4||50||550||100||55||55.25 |- | 3a||48.4||4||50||2420||33.33||80.67|| |- | 3b||5.17||4||50||258.5||333.33||86.17||83.42 |- | 4a||108||4||50||5400||10||54|| |- | 4b||9.56||4||50||478||100||47.8||50.9 |- | 5a||3.3||2||100||330||1||0.33|| |- | 5a||3.2||2||100||320||1||0.32||0.33 |- | 6a||47.9||4||50||2395||10||23.95|| |- | 6b||4.6||4||50||230||100||23||23.48 |- |} Purification table {| {{table}} border = "1" | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"|'''Step''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Purity''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''ssDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''% Recovery''' |- | 1||After first EtOH ppt||1020||9.41||103||105060|| |- | 2||After lambda exon||1103||0.92||55.25||60941||58.01% |- | 3||After qiaquick||420||2.63||83.42||35035||57.49% |- | 4||After USER &DpnII||596||0.7||50.9||26522(*)||75.70% |- | 5||After size selection & Nanosep||4047||0.13||0.33||1315||4.96% |- | 6||After final EtOH ppt||20||202.350||23.48||470||35.70% |- |} # Purification factor: previous step volume/current step volume # % Recovery: previous step mass/current step mass # (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng
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