Qubit® Fluorometer - The Murder of Meredith Kercher
Contents
1. Alterna tively choose Calculate sample concentration to have the Qubit fluorometer perform this calculation for you 4 Record the reading Remove the sample from the instrument insert the next sam ple and press GO 6 Repeat sample readings until all samples have been read 7 If you did not choose Calculate sample concentration cal culate the concentration of your original sample by using the equation supplied below Calculating the Concentration of Your Sample The Qubit fluorometer gives values in either ug mL or ng mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 200 where QF value the value given by the Qubit fluorometer X the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value giv en by the Qubit fluorometer i e if the Qubit fluorometer gave a concentration in g mL the result of the equation will be in g mL Calibration Information The Check Calibration screen displays the raw values for Standard 1 Standard 2 Standard 3 if applicable and the last measurement taken for a given assay These values can as sist you in making a judgment regarding the performance of the Qubit fluorometer and whether or not the samples you used were within ra2. Figure 1 The temperature inside the Qubit fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube remove the tube from the instrument and let it equilibrate to room tempera ture for 30 seconds before taking another reading Calibrating the Qubit Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 2 in General Considerations for Analysis for an example of the calibration curve used to gener ate the quantitation results Assay Tubes for Use with the Qubit Fluorometer Only thin wall clear 0 5 mL PCR tubes are appropriate for use in the Qubit fluorometer Acceptable tubes include Qubit assay tubes Invitrogen Cat no Q32856 500 tubes or Axygen PCR 05 C tubes VWR part number 10011 830 The assay volume must be 200 uL for an accurate read Connecting the Qubit Fluo
3. each assay you have the choice to run a new calibration or to use the values from the previous calibration See Calibrating the Qubit Fluorometer above Highlight either Run new calibration or Use last calibration and press GO Note When using the fluorometer for the first time there will not be an option to Use last calibration Running a New Calibration Calibration of the Qubit fluorometer requires the preparation of the appropriate standard solutions For instructions on the prepa ration of these standards see the instruction manual that accom panies the assay you are using Note It is important to prepare standards in the appropriate 0 5 mL assay tubes Acceptable tubes include Qubit assay tubes 500 Invit rogen Cat no Q32856 or Axygen PCR 05 C tubes VWR part number 1001 1 830 The assay volume must be 200 uL for an accurate read 1 Insert Standard 1 and press GO Ensure you use the Standard 1 appropriate for the assay you are performing 2 The reading will take approximately 5 seconds 3 Insert Standard 2 and press GO Ensure you use the Standard 2 appropriate for the assay you are performing 4 The Quant iT protein assay requires a three point calibra tion so if you are performing this assay a prompt to insert Standard 3 will appear on the screen After Standard 3 is in serted press GO 5 The calibration is complete after Standard 2 is read or after Standard 3 is read for
4. ek E 16 Instrument Specifications 17 Inspection and Setup Upon receiving your Qubit fluorometer please inspect every thing carefully and make sure all accessories are present All ship ments include e Qubit fluorometer e universal power cord with outlet adaptors suitable for use with either a 110V or 220V electrical supply e instruction manual e abbreviated protocol card 1 Position the instrument on a flat dry surface 2 Plug one end of the power cord into the Qubit fluorometer To the other end attach the correct plug adaptor based on the electrical outlet configuration in your country 3 Plug the power cord into the electrical outlet Be sure to use only the power cord provided with your instrument Powering the instrument with an unapproved power supply may dam age the instrument 4 The instrument is automatically powered on when it is plugged in To power down the Qubit fluorometer highlight the Power OFF command on the HOME screen and press GO The Qubit fluorometer has a sleep mode that is triggered af ter 60 minutes of inactivity To recover from the sleep mode press any of the four buttons on the instrument Recovery from sleep mode returns the user to the same screen present when sleep mode commenced After 10 hours of inactivity the unit will power down automatically To recover after pow ering down press any of the four buttons on the instrument Recovery from power
5. the Quant iT protein assay If you get a Standards incorrect message you have made an error Checking the calibration values may help you to determine the source of your error See Calibration Information below Using the Last Calibration You can choose to apply the previous calibration to your sample readings by pressing GO when Use last calibration is highlighted on the calibration selection screen You are then directed to insert the assay tube containing your sample Reading Samples It is important to prepare the sample correctly before a reading is taken For instructions on the preparation of your samples see the instruction manual that accompanies the assay you are using Note It is important to prepare samples in the appropriate 0 5 mL as say tubes Acceptable tubes include Qubit assay tubes 500 Invitro gen Cat no Q32856 or Axygen PCR 05 C tubes VWR part number 1001 1 830 The assay volume must be 200 uL for an accurate read 1 After the calibration is complete or the values from the previ ous calibration have been accepted insert a sample and press GO 2 The measurement will take approximately 5 seconds Upon the completion of the measurement the result will be dis played on the screen The number displayed is the concentra tion of the nucleic acid or protein in the assay tube 3 Calculate the concentration of your original sample see Cal culating the Concentration of Your Sample below
6. Calibration screen on the Qubit fluorometer to confirm that the values for the samples fall between the values of the standards Ensure that the lid is closed while reading standards and samples Prepare samples and standards according to the instructions in the Quant iT assay kit you are using Ensure that the assay is operated entirely at room temperature Standards Incorrect If you get a Standards incorrect message you have made an error Ac cessing the Check Calibration screen will allow you to view the raw val ues for Standard 1 Standard 2 Standard 3 if applicable and the last measurement taken for a given assay These values can assist you in making a judgment regarding the performance of the Qubit fluo rometer and whether or not the samples you used were within range See Calibration Information on page 9 for instructions for accessing the Check Calibration screen Warranty Invitrogen warrants that this product will be free from defects in material and workmanship for a period of one 1 year from date of purchase If a defect is present Invitrogen will at its option repair replace or refund the purchase price of this product at no charge to you provided it is returned during the warranty period This warranty does not apply if the product has been damaged by acci dent abuse misuse or misapplication or from ordinary wear and tear For your protection items being returned must be insured agai
7. Instruction Manual Qubit Fluorometer probes invitrogen com qubit invitrogen Molecular Probes invitrogen detection technologies elcome to the Qubit Quantitation Platform an efficient combination of sophisticated accurate and highly sensitive fluorescence based quantita tion assays with the most user friendly fluorometer available Because the Quant iT assay kits use state of the art dyes selective for dsDNA RNA and protein you don t need to worry about contaminants in your sample affecting the quantita tion And because the Qubit fluorometer uses the very latest in illumination and detection technology for the highest sensitivity you can use as little as 1 ul of sample and still get great results even with very dilute samples So plug in your new Qubit fluo rometer grab your samples and experience higher confidence in downstream applications To get the most out of your new Qubit fluorometer be sure to register online at the Qubit website probes invitrogen com qubit Registering your instrument ensures that you will receive notifications of software upgrades and information on new assays for use with the Qubit fluorometer Table of Contents Inspection and Setup 3 Using the Qubit Fluorometer 5 General Considerations for Analysis 10 Troubleshooting oosa sanaauaaaaa 12 Warranty EE EIERE EELER r EN 14 Obtaining Service 15 Product Listi sags EE oa See
8. Quant iT ssDNA and Quant iT protein assays are generated by a similar algorithm Troubleshooting Handling Samples 1 The calibration standards included in the Quant iT RNA and Quant iT RNA BR Assay Kits are high quality rRNA standards The integrity and concentration of these standards is critical to the op timal performance of the Quant iT RNA assay As such we highly recommend treating the rRNA standards as you would any other precious RNA Use appropriate RNAse free handling techniques in cluding RNAse free gloves pipette tips and tubes Keep the tube lids closed whenever possible do not touch the pipet to the inside wall of the tube when withdrawing a sample and return the rRNA standard to the refrigerator as soon as possible after use Ensure that the assay tubes are at room temperature at the time the reading is taken Do not hold assay tubes in your hand and do not leave assay tubes in the Qubit fluorometer for longer than it takes to read the fluorescence See Effect of Temperature on the Quant iT Assays on page 10 Be careful not to spill sample into the sample chamber Promptly wipe any spills The Quant iT assays are very sensitive and even small amounts of material from a previous sample may result in errors Use a clean 0 5 mL PCR tube for each reading The tube MUST BE CLEAN AND DRY on the outside when taking readings Moisture and condensation on the tube surface can lead to readin
9. eading as this will warm the solution and result in a low reading 80 4 60 4 40 4 Relative fluorescence normalized to room temperature 20 4 0 T T T T 10 15 20 25 30 35 Assay temperature C Figure 1 Effect of temperature on the Quant iT dsDNA BR assay Quant iT dsDNA HS Quant iT ssDNA Quant iT RNA and Quant iT protein assays show similar sensitivities over the same range How the Qubit Fluorometer Calculates Concentration The Qubit fluorometer generates concentration data based on the relationship between the two Standards used in calibration three for the Quant iT protein assay The plot below shows the line corresponding to the curve fitting algorithm a modi fied Hill plot used in the calculation of concentration data for the Quant iT RNA assay For reference the positions of the Standards and a set of data points from an actual experiment are shown su perimposed onto the line This plot demonstrates that the curve fitting algorithm gives accurate values for quantitation N OG Standards o Raw Data Curve Fitting Algorithm Q 1 1 Relative fluorescence D D CO CO i i nm CH L O 100 200 300 400 500 600 Concentration of RNA ng mL Figure 2 The curve fitting algorithm used to determine con centration in the Quant iT RNA assay Data for the Quant iT dsDNA BR Quant iT dsDNA HS
10. ed down mode returns the user to the Welcome screen Registering Your Qubit Fluorometer Visit probes invitrogen com qubit to register your instrument You will be asked to supply the serial number your name and your contact details Registering your instrument ensures that you will receive notifications of software upgrades and information on new assays for use with the Qubit fluorometer Installing the Qubit Data Logger The Qubit fluorometer is designed for stand alone use and does not require a connection with a PC If data transfer to your PC is de sired you will need to install the Qubit Data Logger Visit the web address below and follow the instructions When the software is installed the Qubit Data Logger icon will appear on your desk top The requirements for download are USB A mini B 5 pin cable available from Invitrogen Cat no Q32858 or from most of fice supply retailers PC System requirements USB port 1 1 or later e Windows XP Location of downloadable software probes invitrogen com qubit Using the Qubit Fluorometer Critical Quant iT Assay Considerations Assay Temperature The Quant iT assays were designed to be performed at room temperature 22 28 C as temperature fluctuations can influence the accuracy of the assay Figure 1 on page 10 To minimize tem perature fluctuations store all kit reagents at room temperature and insert all assay tubes into the Qubit fluoro
11. efore repairing the unit Repair and ship ping costs both ways are your responsibility if the instrument is not under warranty If you are outside the United States and have purchased the in strument from an authorized Invitrogen distributor contact that distributor directly If you purchased directly from Invitrogen you are welcome to send the instrument for free repair within the lim its of the warranty using the procedure described above If it is not under warranty we will repair it on a time and materials basis We cannot however pay for shipping duties and documentation costs outside the continental United States Product List Cat no Qubit Fluorometer Qubit fluorometer Q32857 Qubit Starter Kits Qubit Quantitation Starter Kit includes 1 Qubit 032860 fluorometer and 5 Quant iT assay kits Qubit Quantitation Lab Starter Kit includes 5 Qubit 032861 fluorometers and 5 Quant iT assay kits Quant iT Assay Kits for Use with the Qubit Fluorometer Quant iT dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit fluorometer Q32850 Quant iT dsDNA BR Assay Kit 500 assays 2 1000 ng 032853 for use with the Qubit fluorometer Quant iT dsDNA HS Assay Kit 100 assays 0 2 100 ng 032851 for use with the Qubit fluorometer Quant iT dsDNA HS Assay Kit 500 assays 0 2 100 ng 032854 for use with the Qubit fluorome
12. g errors Minute bubbles in samples will cause errors in readings Be sure not to introduce bubbles into samples Slight tapping on the tube wall or brief centrifugation will often help dissipate bubbles Low Reading 1 Your sample is out of range Use a sample that is more concentrated or use a lower dilution i e 20 uL in 180 uL instead of 10 uL in 190 uL View the Check Calibration screen on the Qubit fluorometer to confirm that the values for the samples fall between the values of the standards see Calibration Information on page 9 Ensure that you have prepared the Quant iT working solution cor rectly 1 200 dilution using the buffer provided in the kit Ensure that you have prepared the standard tubes correctly 10 uL of each standard in 190 uL of Quant iT working solution Ensure the tubes are filled to at least 200 uL standards and sam ples Ensure that the reagents and standards you are using are not older than the expiration dates assigned by the manufacturer The Quant iT stock solution should be protected from light as much as possible Select the correct Qubit fluorometer assay for the Quant iT assay you are performing and calibrate the Qubit fluorometer correctly Standards must be used in the correct order Ensure that the assay is operated entirely at room temperature High Reading 1 2 Your sample is out of range Use a sample that is less concentrated View the Check
13. meter only for as much time as it takes for the instrument to measure the fluores cence as the Qubit fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time To allow the Quant iT assay to reach maximum fluorescence incu bate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incuba tion period the fluorescence signal is stable for 3 hours at room tem perature for all nucleic acid assays except the Quant iT ssDNA as say which is stable for up to 30 minutes The Quant iT protein assay requires 15 minutes incubation for a stable signal For greatest accu racy of the protein assay the incubation time of the samples should be within 10 minutes of the incubation time of the standards Photobleaching of the Quant iT Reagent The Quant iT reagents exhibit high photostability in the Qubit fluorometer showing lt 0 3 drop in fluorescence after 9 read ings and lt 2 5 drop in fluorescence after 40 readings It is impor tant to remember however that if the assay tube remains in the Qubit fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in tem perature see General Considerations for Analysis
14. nge For the Quant iT dsDNA BR Quant iT dsDNA HS Quant iT ssDNA and Quant iT RNA assays the reading given by Standard 2 should be much higher than that of Standard 1 and the reading of your sample should fall somewhere between that of the two Standards For the Quant iT protein assay the reading given by Standard 3 should be much higher than that of Standard 2 and the reading of your sample should be no more than 50 greater than Standard 3 1 To access the Check Calibration screen press HOME from any screen 2 Use the Aor V key to highlight the Check Calibration option and press GO 3 On the Check Calibration screen use the A or V key to select the assay for which you want calibration information and press GO 4 The raw values will be displayed on the screen General Considerations for Analysis Effect of Temperature on the Quant iT Assays The Quant iT assays were designed to be performed at room temperature 22 28 C as temperature fluctuations can influence the accuracy of the assay Figure 1 To minimize temperature fluc tuations store all kit reagents at room temperature and insert all assay tubes into the Qubit fluorometer only for as much time as it takes for the instrument to measure the fluorescence as the Qubit fluorometer can raise the temperature of the assay solu tion significantly even over a period of a few minutes Do not hold the assay tubes in your hand before r
15. nst possible damage or loss Invitrogen cannot be responsible for damage incurred during shipment of a repair instrument It is recommend that you save the original packing material in which the instrument was shipped This warranty shall be limited to the replacement of defective products IT IS EXPRESSLY AGREED THAT THIS WARRANTY WILL BE IN LIEU OF ALL WARRANTIES OF FITNESS AND IN LIEU OF THE WARRANTY OF MERCHANT ABILITY Obtaining Service Contact Invitrogen Technical Service in the USA and Canada call 1 800 955 6288 or send an email to probestech invitrogen com and describe the problem s you are experiencing Carry out any suggested modifications or tests DO NOT ship an instrument to us without prior telephone or email contact Often the problem is a rel atively simple one that you can solve yourself with our direction If it is determined by the Invitrogen Technical Service representa tive that the instrument should be returned for repair a Decon tamination Certificate will be faxed to you Once you have com pleted the certificate signed it and returned it to the Invitrogen Technical Service representative a Return Authorization number will be assigned and you will receive instructions for the return If you are in the United States and the instrument is under war ranty Invitrogen will repair or replace the unit and pay for return shipment If the instrument is not under warranty Invitrogen will give you a cost estimate b
16. rometer to PC for Data Transfer Optional It is not necessary to connect the Qubit fluorometer to a PC Quantitation results are displayed directly on the screen for you to record If data transfer is desired you need to have 1 Qubit Data Logger installed on your PC see Installing the Qubit Data Logger above 2 Qubit fluorometer connected to the PC via the USB A mini B 5 pin cable The Qubit fluorometer should be connected to the PC before the Qubit fluorometer is powered on When the Qubit fluorometer is powered on the PC may then let you know that it has recog nized a peripheral device As you proceed through the assay the open spreadsheet will be updated with the data for standards and samples data transfer is initiated when the first standard is read At the end of the experiment don t forget to save the spreadsheet that contains the data for that session Selecting a Quant iT Assay 1 If the fluorometer is powered down or in sleep mode press any of the four buttons to power up the fluorometer Note If the instrument was in sleep mode pressing any of the four buttons returns you to the last screen viewed before sleep mode commenced To get to the HOME screen after reawakening press HOME 2 On the HOME screen use the A or V key to highlight the as say of choice 3 When the Quant iT assay you would like to run is highlight ed press GO Calibrating the Qubit fluorometer For
17. ter Quant iT ssDNA Assay Kit 100 assays 1 200 ng for 010212 use with the Qubit fluorometer Quant iT RNA Assay Kit 100 assays 5 100 ng 032852 for use with the Qubit fluorometer Quant iT RNA Assay Kit 500 assays 5 100 ng 032855 for use with the Qubit fluorometer Quant iT RNA BR Assay Kit 100 assays 20 1000 ng 010210 for use with the Qubit fluorometer Quant iT RNA BR Assay Kit 500 assays 20 1000 ng 010211 for use with the Qubit fluorometer Quant iT Protein Assay Kit 100 assays 0 25 5 ug 033211 for use with the Qubit fluorometer Quant iT Protein Assay Kit 500 assays 0 25 5 ug 033212 for use with the Qubit fluorometer Qubit Accessory Products Qubit assay tubes 500 tubes Q32856 Qubit USB cable Q32858 Qubit international power cord replacement Q32859 Instrument Specifications Specification Description Size 4 5 x 6 5 x 1 8 11 4 cm x 16 6 cm x 4 6 cm Weight 7 7 oz 219 g Dynamic range 5 orders of magnitude Operating temperature 59 98 F 15 40 C Light sources Blue LED max 460 nm Red LED max 635 nm Excitation filters Blue 400 490 nm Red 570 645 nm Emission filters Green 510 580 nm Red 655 725 nm Detectors Photodiodes measurement capability from 300 1000 nm Calibration type 2 or 3 point standard Tube type 500 uL clear tubes Warm up time 2 seconds in
18. vitrogen Molecular Probes www invitrogen com invitragen detection technologies 2007 Invitrogen Corporation All rights reserved These products may be covered by one or more Limited Use Label Licenses see Invitrogen catalog or www invitrogen com By use of these products you accept the terms and con ditions of all applicable Limited Use Label Licenses For research use only Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated 0 070233 r4 US 101507
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