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Amplification Curves

Problem

Cause

Solution

Amplification in
No Template
Control (NTC)

a. Reagent or genomic
contamination

Repeat protocol with fresh
reagents

b. Primer-dimer
formation

Use a master mix with good
hot start activity

Looping Data
Points during
early cycles; noisy
curve in early
cycles

a. Baseline adjustment
contains too many cycles

Reset baseline to 3 cycles
before indication of
amplification

b. Too much starting
material

Use less starting material

Slope of curve is
greater than or
less than -3.34
and R^2 is less
than 0.98

a. Inaccurate dilutions

Recalculate standard
concentration or gene copy
number using a
spectrophotometer; make
new stock solutions of the
control standards; eliminate
extreme concentrations

b. Standard curve exceeds
the linear range of
detection

Plateau has lower
than expected
fluorescence units

a. Limiting reagents

Confirm master mix
calculations

b. Degraded reagents (i.e.
master mix or dNTPs)

Repeat protocol with fresh
reagents

Содержание Open qPCR

Страница 1: ...Open qPCR User Manual Revision A Open qPCR User Manual Research use only ...

Страница 2: ... instrument Research use only Not for use in diagnostic procedures DISCLAIMER All examples in this document are for information and illustration purposes only TRADEMARKS Products that are referred to in this document may be either trademarks and or registered trademarks of the respective owners Chai makes no claim to these trademarks ChaiTM is a trademark of Chai Biotechnologies Inc SYBRâ is a reg...

Страница 3: ...s USB Ethernet Wi Fi 2 3 Setting a Static IP Address 2 4 Logging in and Creating an Account CHAPTER 3 Calibration 3 1 When to Calibrate the Open qPCR 3 2 Adjusting the Open qPCR Lid Height 3 3 Open qPCR Single Channel Calibration 3 4 Open qPCR Dual Channel Calibration CHAPTER 4 Assay Setup 4 1 General PCR Considerations 4 2 Create A New Experiment 4 3 Starting Cancelling Runs CHAPTER 5 Result Anal...

Страница 4: ... 6 2 Software Update 6 3 Network Settings 6 4 Diagnostics CHAPTER 7 Maintenance 7 1 Cleaning and Disinfecting 7 2 Open qPCR Return and Repair CHAPTER 8 Troubleshooting 8 1 Amplification Curves 8 2 Melt Curves 8 3 Factory reset Appendix A Glossary ...

Страница 5: ...vant hazards and or important information regarding operation of the instrument Icon Definition CAUTION Indicates that you should consult the manual for further information and proceed with appropriate caution Failure to do so may lead to physical injury or could cause damage to the instrument ELECTRIC SHOCK HAZARD Indicates the presence of an electric shock hazard and to proceed with appropriate ...

Страница 6: ...ak in the electrical ground path may create a hazardous condition Do not touch the power switches or power cord with wet hands Always power down the instrument and disconnect the power cord before performing cleaning or maintenance procedures The heat block and lid temperature rises above 95 C To avoid injury do not touch the heat block lid or tubes immediately after a run Let the surface cool for...

Страница 7: ...s are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause...

Страница 8: ...hooting help on this site Community Forum Site Please visit https community chaibio com to connect with the global community regarding Chai products application uses and general tips on PCR For instrument purchases made through a distributor please contact your local support service representative for assistance For instrument purchases made directly through Chai please contact the Chai support st...

Страница 9: ...et allowing simultaneous analysis for as many samples as desired Chai offers two models of the Open qPCR system single channel and dual channel The two instruments differ primarily in the number of fluorophores each can detect The single channel detects either SYBR Green Chai Green or FAM fluorophores through channel 1 whereas the dual channel allows for additional detection of HEX VIC or JOE fluo...

Страница 10: ... rate 5 0 C s Min ramp rate 0 00001 C s Design dual Peltiers Temperature accuracy 0 2 C at 65 C s Temperature uniformity 0 4 C at 65 C s Lid temperature Ambient to 120 C Additional Notes Allowed supply voltage fluctuations up to 10 of the nominal voltage Average power rating tested at room temperature with default protocol 1 3 General Features 1 Adjustable lid knob 2 Lid lever 3 Lid latch 4 LCD to...

Страница 11: ...pposite side can then be cooled and heated with respect to the heat sink Figure 1 3 Figure 1 3 A copper heat spreader beneath the Peltiers ensures temperature uniformity and rapid heat transfer during cycling Two thermistors attached directly to the heat block monitors well temperature Cooling is achieved with a high efficiency fan that dissipates air across the heat sink Reaction wells Fan Heat s...

Страница 12: ... Figure 1 4 depicts all components involved in the optical detection Figure 1 4 During a run the LED emits light through an excitation filter embedded within the heating block on the instrument lid The incident light strikes the top of each reaction tube and excites the fluorophores in the sample The emerging light then passes through the emission filters before being detected by the photodiode bo...

Страница 13: ... Consumables The instrument utilizes 100 µL low profile PCR tube strips Due to Open qPCR s unique architecture both cap and tube sides must be optically clear and they must not auto fluoresce For optimal results please use Chai s validated DNAse and RNAse free tubes Table 1 7 Product Catalog PCR 8 Cap Strips Optically Clear Flat Caps 100 µL qty 250 S02112 PCR Tube Cap Strips 8 Well Strips Opticall...

Страница 14: ...ering the instrument Connect the power cable from the power source to the back of the instrument before turning on the power switch After the switch is turned on the LCD screen will display Booting on the bottom right corner during initialization Give the instrument approximately five minutes to boot up Ensure that the instrument s software version and serial number are displayed on the LCD screen...

Страница 15: ...at your computer may require a systems administrator to grant access permission prior to install 2 Once the appropriate drivers are installed make sure the instrument is already powered on and then connect the USB cable from your computer to the USB port on the back of the instrument 3 After the system has been turned on and running for approximately 2 3 minutes open an internet browser and type i...

Страница 16: ...ent Settings Figure 2 1 Make sure this IP address is also displayed on the bottom left of the instrument s LCD screen Figure 2 1 7 Unplug the USB cable and close out of the 192 168 7 2 browser Open a new browser and type in the instrument IP address to access the software 2 3 Setting a Static IP Address The Open qPCR system must be connected by Ethernet in order to set a static IP address If appli...

Страница 17: ...on both the current web browser and the instrument s LCD screen 2 4 Logging in and Creating an Account Navigate to the appropriate address in your web browser to view the following screen Figure 2 3 Proceed to create a new user account for logging into the software If the instrument is connected by Ethernet or Wi Fi different users may log into the software simultaneously to view previous test res...

Страница 18: ... PCR tubes or individual 100 µL PCR tubes Use gloves while handling the tubes as prints on the tubes may affect the fluorescence measurements Ensure there are 16 capped tubes in the heat block during the lid calibration process Raise the lid height by turning the knob on the lid clockwise Place the PCR tubes in the instrument and then close the lid until it latches Proceed to turn the knob counter...

Страница 19: ...n If the lid height is adjusted a re calibration is required PCR tubes used for the calibration process must be the same tubes that will be used for subsequent test runs Fluorescein Calibrator Kit Table 3 2 Fluorescein Calibrator of Calibrations Quantity Catalog Shipped with instrument 3 1 x 1 3 mL vial Available for order 6 2 x 1 3 mL vial S0110M Storage Temperature Store at 4 C or 20 C long term...

Страница 20: ...repare the water strips by aliquoting 25 µL tube of water into 2 new PCR strip tubes Cap strip tubes tightly and spin down Calibration Procedures Please note that the calibration process must be completed in one sitting The heat block and lid temperature rises above 95 C To avoid injury do not make direct contact with the heat block lid or tubes immediately after a run Let the surface cool for a f...

Страница 21: ...ure 3 4 Gather the previously prepared strip solutions and select Begin Figure 3 4 5 Insert two water strips into the instrument Press down on the tubes gently once they are inserted Adjust the lid height per the instructions in section Adjusting the Open qPCR Lid Height Figure 3 5 ...

Страница 22: ...lid heating and data collection process Figure 3 6 7 Allow the instrument to read fluorescence for 2 3 minutes before proceeding to the next step Figure 3 7 8 Once the reading completes insert the Fluorescein solution strips Figure 3 8 Press down on the tubes gently once they are inserted Click Next Figure 3 8 ...

Страница 23: ...se the calibration dyes because photo bleaching occurs at different levels from well to well If you would still like to reuse the dyes pool the solution first and aliquot again as before 3 4 Open qPCR Dual Channel System Calibration The Open qPCR dual channel system requires calibration with the FAM HEX Calibrator Set The calibrators are provided in lyophilized format Prior to calibrating the Open...

Страница 24: ...0 nM and 1000 nM respectively Storage Temperature Store the lyophilized calibrators and the reconstitution buffer between 4 20 C The calibrators are light sensitive Ensure that the vials are placed in a dark location Storage temperature of the reconstituted calibrators should be at 4 C and protected from light When stored at the recommended temperature and condition the lyophilized calibrator and ...

Страница 25: ...incubation open the vial caps and use a new tip to pipette up and down ten times or vortex for 10 seconds 5 Spin down the vials using a mini centrifuge to collect all liquid contents at the bottom of the tube The reconstituted calibration vials are now ready for use Preparing for Calibration The FAM and HEX solutions are light sensitive Keep aliquoted solutions protected from light If you are usin...

Страница 26: ... removing the tubes If samples must be removed immediately avoid contact with surfaces by extracting tubes with tweezers Always wear gloves and protective eye wear when handling reagents or operating the instrument 1 Log into your account through a web browser On the software home screen navigate to Settings Calibration 2 Under Optical Calibration select Run Now 3 The calibration time and material...

Страница 27: ...he tubes gently once they are inserted Adjust the lid height per the instructions in section Adjusting the Open qPCR Lid Height Figure 3 14 6 Close the lid making sure that the latch clicks and select Next You will see the screen below during the lid heating process Figure 3 15 ...

Страница 28: ...igure 3 16 8 Once the reading completes insert the FAM strips Figure 3 17 Figure 3 17 9 Repeat steps 6 7 for both the FAM and HEX strips 10 Following the subsequent fluorescence reading you will receive a status displaying Calibration Complete Figure 3 18 You may now proceed to your experiment setup ...

Страница 29: ...libration process in such instances If failure persists on repeat contact Technical Support Figure 3 19 It is not recommended to reuse the calibration dyes because photo bleaching occurs at different levels from well to well If you would still like to reuse the dyes pool the solution first and aliquot again as before ...

Страница 30: ...tips and change tips after each use o Ensure that reaction and reagent components are always capped when not in use o Clean lab benches and equipment periodically with 70 ethanol or 10 bleach followed by 70 ethanol 4 2 Create a New Experiment To set up a new assay procedure log into your account through the appropriate web address On the home screen click on the green icon titled Create A New Expe...

Страница 31: ...between a range of 0 00001 and 5 C s If no ramp speed is designated the system defaults to Auto Auto equates to the maximum ramp speed on the instrument Figure 4 4 INFINITE HOLD This feature is available only during the last step with Gather Data turned off To implement an infinite hold set the Hold Duration to 00 00 and press Enter on your keyboard The infinity symbol will appear next to the temp...

Страница 32: ... with a holding stage that contains the initial denaturation step Additional steps may be added to a holding stage if needed Generally in both two step and three step PCR protocols a holding stage exists at the very beginning since it houses the initial denaturation step In some cases depending on whether you plan to run downstream applications such as a post PCR agarose gel an additional holding ...

Страница 33: ...experiment Figure 4 9 Figure 4 9 AUTODELTA The Autodelta option is used for Touchdown PCR TD PCR and can be applied only to the cycling stages on the protocol setup page Touchdown PCR can be incorporated as part of any standard PCR to increase the specificity sensitivity and yield of the PCR reaction It increases the likelihood that your target of interest is amplified in cases where there may be ...

Страница 34: ...ed on the right hand side To delete a step select the Ä icon To reposition a step in the protocol click and drag the dotted icon at the bottom of the orange section Figure 4 12 ENDPOINT REACTIONS For end point reactions with no fluorescence detection conventional PCR make sure to turn off Gather Data for Step and or Ramp in all stages of your protocol No amplification curves will be displayed on t...

Страница 35: ... time remaining on the experiment is shown on the top right corner To abort the current run click on the icon on the bottom right corner 3 While the experiment is running you may access other screens without aborting the test To do so click on the icon located at the top left near the experiment name You will have options to go back to the home page or view the experimental details Under the Exper...

Страница 36: ...otted against the cycle number The analyzed data is expressed as a numerical representation Cq values that is displayed on the right side of the page palette These Cq values are inverse to the amount of target nucleic acid present in a given reaction A low Cq value indicates high quantities of target nucleic acid whereas a high Cq value indicates minimal target quantities Figure 5 1 AMPLIFICATION ...

Страница 37: ...the first derivative of the curve must exceed for a Cq to be called Min 2nd Derivative The threshold which the second derivative of the curve must exceed for a Cq to be called Baseline Cycles Baseline refers to the background signal level prior to any significant product amplification The baseline should be wide enough to eliminate any background found in early cycles of amplification Auto Allows ...

Страница 38: ...results screen of a completed sample run in a dual channel cycler To view the exact Cq and Relative Fluorescence Units RFU for a specific cycle click on one of the curves Figure 5 5 Figure 5 4 Figure 5 5 SAMPLE NAME INPUT Sample names may be entered in the software post run for tracking purposes Once the run has completed select each box under the Sample Name column and type in the sample name as ...

Страница 39: ...r Linear format There is no option for differentiating between channels since there is only one channel 5 2 Melt Curve MELT CURVE ANALYSIS The Melt Curve feature may be used as a quality control for cycling analysis The purpose of a melt curve analysis is to determine the peak dissociation temperature of the resulting amplicons This aids in measuring an assay s analytical specificity such as forma...

Страница 40: ...l to the amount of double stranded target DNA strand dissociation results in decreased fluorescence intensity The inflection point seen in the sigmoidal shaped curve below equates to the melting temperature Tm of the amplified product Figure 5 9 Derivative Plot This is a plot of the negative first derivative of the normalized curve Each peak is characteristic for a specific amplicon The Tm is easi...

Страница 41: ...e 5 11 The thermal profile graph displays time on the x axis and heat block temperature on the y axis You can manually hover over the graph to view the heat block and lid temperature at each time point Figure 5 12 5 4 Navigation Icon on Results Screen To navigate out of the results screen select the icon at the top left of the page You will then see options as shown in Figure 5 13 Figure 5 13 ...

Страница 42: ...cation runs contain the following files amplification cq and temperature_log To determine the absolute copy number from the starting material use the cq csv file to generate the standard curve o amplification csv contains the baseline subtracted background subtracted and sample fluorescence values for each corresponding channel well and cycle number o cq csv provides the Cq values for each channel...

Страница 43: ...ft of the results screen Proceed to click on the experiment name Figure 5 15 to delete before typing in the new name Figure 5 15 5 7 Deleting Experiments To delete previous test runs click on the Edit tab in the Experiments column on the home screen Once you select Edit you will see the icon next to each test Click on it to delete the respective run Select Edit to return to the original screen Fig...

Страница 44: ...instrument is not on the most current version the software will automatically prompt you to update once logged in This prompt occurs only when the instrument is connected by Ethernet or Wi Fi The update process will take approximately 30 60 minutes Ethernet or Wi Fi If the Open qPCR is connected to the local network the software update image will be displayed as follows Figure 6 3 Figure 6 3 USB I...

Страница 45: ... have internet access via an instrument or computer please contact Technical Support 6 3 Network Settings You may connect your Open qPCR instrument to any available network via Ethernet or Wi Fi Ensure that your computer is connected to the same network as the instrument You have the option to adjust your Ethernet settings as necessary under the Network Settings tab Refer to Section 2 2 Connectivi...

Страница 46: ...e diagnostic tests the lid heating as well as the heating and cooling efficiency on the processing block Once completed you will see the following screen Figure 6 7 A pass is indicated by a green check mark for the specific tested component Figure 6 7 Optical Diagnostic The optical diagnostic checks for functionality of the fluorescence detection optics The single channel model does not require an...

Страница 47: ...47 Figure 6 8 Dual Channel Figure 6 9 Single Channel ...

Страница 48: ...s o Do not flood the instrument with the cleansing solution o Do not spray the cleaning agent directly onto the instrument as this could damage internal components o Spray the cleaning agent on low lint cloth or paper towel and gently wipe down all surfaces o To clean the wells of the PCR instrument use a low lint cotton swab dipped in 70 ethanol Avoid using bleach if possible This is corrosive an...

Страница 49: ...ce it must be disinfected prior to shipment For direct customers of Chai please contact Chai Technical Support for a copy of the Decontamination Certificate to initiate the return process For international customers please contact your local supplier for the instrument repair process ...

Страница 50: ...ber below the LOD for the assay Increase sample concentration c Pipetting errors Repeat assay 2 No amplification a Lack of target in sample Confirm by testing a positive control sample b Assay design failure Try a different assay c Sample degradation For RNA samples denature via formamide and then run an agarose gel For DNA samples run an agarose gel or use a capillary electrophoresis system A sme...

Страница 51: ...ches correctly 3 No real time curve and no visible bands on gel a Master mix preparation error Confirm master mix calculations b Protocol requires optimization Test different annealing temperatures and times c Sample component inhibiting PCR Dilute sample or change extraction procedure d Excess fluorescent dye inhibiting PCR Decrease fluorescent dye concentration e Degraded PCR reagent Rerun proto...

Страница 52: ...cy has decreased over time by re running a previously working assay If not assay specific confirm proper storage of master mix and or template Instrument Confirm that the cycling temperatures and time parameters are correctly set b Master mix differences Test different master mixes with the same protocol assay design and reaction components Confirm assay reproducibility c Sample component inhibiti...

Страница 53: ...tube type used Use Chai s validated PCR strip tubes The instrument requires 100 µL optically clear non auto fluorescent tubes 7 Very low fluorescence signal a Low copy number of template Increase concentration or add more volume of sample b High background signal Decrease fluorescent dye or probe concentration Check probe purity c Sample component inhibiting PCR Dilute sample or change extraction ...

Страница 54: ...dication of amplification b Too much starting material Use less starting material 10 Slope of curve is greater than or less than 3 34 and R 2 is less than 0 98 a Inaccurate dilutions Recalculate standard concentration or gene copy number using a spectrophotometer make new stock solutions of the control standards eliminate extreme concentrations b Standard curve exceeds the linear range of detectio...

Страница 55: ... Run the sequence through Primer BLAST to check for homology d Excessive hold times Reduce the number of hold times 8 3 Factory Reset A factory reset will restore your machine to factory condition Remember to back up your data before performing a reset as this procedure will erase all data To perform the factory image reset 1 Turn off your Open qPCR Disconnect any USB cables 2 Using a toothpick pr...

Страница 56: ... for about 30 minutes After 30 minutes the device will reboot and the Open qPCR logo will be displayed on the LCD 5 The device is now configured in the manner it was when you first received it You may now reconnect USB cables and create a new account Additionally you should update your software to the latest version as it will be downgraded by the factory reset procedure ...

Страница 57: ... in the sample A high Cq value equates to a low quantity of the target sequence in the sample Cycling Stage Also termed as the amplification stage this refers to a stage that is repeated in the thermal profile setup FAM A carboxyfluorescein fluorescent dye with lmax absorption at 494 nm and lmax emission at 518 nm HEX A hexachlorofluorescein fluorescent dye with lmax absorption at 535 nm and lmax ...

Страница 58: ... PCR This type of PCR contains a single primer set in the tube well where only target or endogenous control can be amplified at a time VIC A fluorescent dye with lmax absorption at 538 nm and lmax emission at 554 nm ...

Страница 59: ...59 Corporate Headquarters 990 Richard Ave Suite 110 Santa Clara CA 95050 Toll Free 1 800 642 4002 International 1 650 779 5577 www chaibio com ...

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