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Agilent Technologies SureCycler 8800, Setup And User Manual

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Agilent Technologies SureCycler 8800 Setup And User Manual Download Page 58

4

PCR  Programs

 

Troubleshooting

58

Agilent SureCycler 8800 Setup and User’s Guide

Troubleshooting

This section describes potential problems and recommended actions.

If PCR yield is low when running small reaction volumes with the 
Standard ramp rate

✔

If you are using sealing films/mats, shorten the duration of the 
denaturation step during cycling. Denaturation times as short as 3–5 
seconds may be appropriate. Alternatively, switch to using capped 
reaction tubes. 

✔

Increase the reaction volume to 50 

μ

l.

✔

Try switching to the Fast ramp rate. 

If no actual temperature is displayed during the run

The thermal block unit may not be correctly seated on the instrument. 

✔

Check that the unit is installed correctly and reboot the cycler.

If the program does not start

The thermal block unit may not be correctly seated on the instrument. 

✔

Check that the unit is installed correctly and reboot the cycler.

Summary of Contents for SureCycler 8800

Page 1: ...Agilent Technologies SureCycler 8800 Setup and User s Guide Version A March 2011...

Page 2: ...ate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of su...

Page 3: ...n instructions for setting up the cycler instrument 3 Getting Started This chapter contains instructions to load samples get started with the software set up user accounts and access instruments remot...

Page 4: ...4 Agilent SureCycler 8800 Setup and User s Guide...

Page 5: ...stall the thermal block on the instrument base 16 Step 4 Connect the instrument to a power supply 17 Step 5 Connect optional devices 17 Step 6 Turn on the instrument 17 Step 7 Connect the cycler to a...

Page 6: ...Cycler Settings 32 Hot Top Setting 32 Cycler name 33 Time and date 33 4 PCR Programs 35 Creating New PCR Programs 36 To manually create programs 36 To create a PCR program using the PCR Wizard 39 Tip...

Page 7: ...Troubleshooting 58 If PCR yield is low when running small reaction volumes with the Standard ramp rate 58 If no actual temperature is displayed during the run 58 If the program does not start 58 If c...

Page 8: ...8 Agilent SureCycler 8800 Setup and User s Guide Contents...

Page 9: ...re You Begin Materials Provided 10 Safety Precautions 10 Indicator LED Light 13 Overview 13 SureCycler 8800 Technical Support 14 This chapter contains information for you to read and understand before...

Page 10: ...urn on the instrument for the first time The device must be connected to a grounded socket Do not touch any switches or outlets with wet hands Turn off the instrument before you disconnect the power c...

Page 11: ...block inner side of heated lid and reaction vessels These areas quickly attain temperatures of greater than 50 C Keep the heated lid closed until temperatures of 30 C or lower are reached Do not use a...

Page 12: ...interfere with the normal operation of the USB ports on the instrument Handling precautions are required when working in high static environments Wear a grounded wrist strap and take other antistatic...

Page 13: ...e most complex thermal cycling techniques including time and temperature increments touchdown PCR and temperature gradients The software interface operated through the color touchscreen features an in...

Page 14: ...services agilent com World Wide Web www genomics agilent com Telephone USA and Canada 800 227 9770 Europe Japan 0120 477 111 All other countries Go to www genomics agilent com and click on the Contact...

Page 15: ...e thermal block on the instrument base 16 Step 4 Connect the instrument to a power supply 17 Step 5 Connect optional devices 17 Step 6 Turn on the instrument 17 Step 7 Connect the cycler to a network...

Page 16: ...pen the shipping containers and unpack the contents Detailed unpacking instructions are provided on the installation poster packed inside the same box as the instrument base Step 3 Install the thermal...

Page 17: ...ional USB device such as a keyboard mouse or memory stick to the instrument via the USB ports on the front of the instrument 1 Plug the USB cable of the device into either USB port on the instrument S...

Page 18: ...4 With the buttons firmly pressed lift the thermal block unit up and off the instrument base Use care when lifting the thermal block unit to avoid letting the lid fall closed on your hands Keeping th...

Page 19: ...User Accounts 24 Access Levels 24 Logging In to User Accounts 24 Managing User Accounts 26 Network Settings and Remote Access 29 To view or edit the cycler s IP address and name 29 To access the cycl...

Page 20: ...tion plate use a compression mat during cycling to ensure the film stays firmly adhered to the plate See Agilent Plasticware and Consumables on page 21 for ordering information on a compression mat To...

Page 21: ...The following PCR plates reaction tubes and sealing films are compatible with the SureCycler 8800 Agilent Part Number Description 401333 96 well plate 25 pack 410188 384 well plates 50 pack 410186 Adh...

Page 22: ...point for all the software controlled instrument operations as well as file and account management Each navigation button on the Home screen menu takes you to a different functional area in the softw...

Page 23: ...nt SureCycler 8800 Setup and User s Guide 23 Login Opens the Login screen Settings Use this button to adjust cycler settings upgrade software and manage user accounts Table 3 Home Screen Button Descri...

Page 24: ...In to User Accounts To log in as Guest No password is required to login as Guest By default the Guest account is automatically logged into the system each time the instrument is turned on Table 4 Acce...

Page 25: ...ect Administrator from the Username drop down list and enter the password The default Administrator password is ADMIN Use the Shift key on the virtual keyboard to toggle between lowercase and uppercas...

Page 26: ...d for that account If logging in as Guest leave the Password text box blank 4 Click or touch the Login button A confirmation message appears to verify the name of the user logged in to the system Mana...

Page 27: ...field enter a user name 5 In the Password and Confirm Password fields enter a password for the account 6 Select the access level User or Administrator from the Access Level drop down list Selecting Us...

Page 28: ...ccount Note that the access level for the Administrator and Guest accounts cannot be edited 5 Press OK to save the changes A confirmation message appears after successful update of the user data To de...

Page 29: ...and the DHCP server You have the option of obtaining an IP address automatically or assigning one manually If you select the option Obtain an IP Address automatically the fields for network informatio...

Page 30: ...me of the instrument you want to connect to into the IP Address Cycler field 4 Press Connect You will be returned to the Network Control screen and the network information and status of the connected...

Page 31: ...access the cycler from a PC 1 From the PC open an internet browser application Microsoft Internet Explorer is recommended 2 In the URL field enter the IP address for the cycler The SureCycler 8800 so...

Page 32: ...ting screen make a selection for use of the hot top Select Yes to use the hot top when the instrument is running Select No to turn the hot top off 4 Confirm your selection by pressing OK When the hot...

Page 33: ...ress System Setting 3 Press the Change button located near the top of the screen A new screen will open prompting you to enter a new cycler name 4 Confirm your changes by pressing OK Time and date The...

Page 34: ...d Time and date 34 Agilent SureCycler 8800 Setup and User s Guide 4 To change the time press the up down arrows to adjust the hour minutes or seconds listed under System Time 5 Confirm your changes by...

Page 35: ...PCR Wizard 39 Tips for Creating PCR Programs for the 384 well SureCycler 8800 42 Program Steps in the Commands List 43 Using a Preloaded PCR Program 51 Editing PCR Programs 53 Running a PCR Program 5...

Page 36: ...program automatically The two methods are described in the following sections To manually create programs Programs can be created manually from the programming screen The steps in the Command List ar...

Page 37: ...e first step of the program from the Command List to the Program Window using one of the following two approaches Press the command then press the forward arrow button shown below Double press the com...

Page 38: ...e Save Program screen will open b Press the keyboard icon shown below left to access the touchscreen s virtual keyboard and use the keyboard to type a name into the Program Name field Once the name ha...

Page 39: ...of the PCR Wizard 1 Polymerase Selection Select a DNA polymerase from the list and press Next The polymerase options are Paq5000 hot start or standard version PfuUltra II Herculase II PfuTurbo hot st...

Page 40: ...s If you entered the base sequences of the primers rather than the Tm s the software calculates the Tm for each primer based on the formula Tm 69 3 0 41 GC 650 N where GC is the percentage of GC conte...

Page 41: ...riate gradient range based on the melting temperatures of the primers Once the wizard has guided you through these five steps it creates a PCR program based on the provided information and displays it...

Page 42: ...well block reducing the duration of the denaturation step during cycling may improve product yield for certain enzymes Denaturation times as short as 3 5 seconds may be optimal in some protocols If y...

Page 43: ...es with hot start technology The extended denaturation step activates the polymerase by either removing a chemical modification agent or by denaturing a polymerase bound antibody The Hot Start command...

Page 44: ...Step Enter the denaturation temperature in C left field and denaturation time in hours minutes seconds right field Annealing Step Enter the annealing temperature in C left field and annealing time in...

Page 45: ...before a series of steps that are to be repeated for several cycles Number of Cycles Enter the number of times the series of steps should be cycled Cycle Name Optional If desired enter a user selecte...

Page 46: ...er the temperature for the step in C Hold for Enter the duration of the step Units are hours minutes seconds Temperature Ramp Select the ramp rate for the instrument The Fast ramp rate is the default...

Page 47: ...Start Cycle and an End Cycle command Insert this program step to terminate a PCR cycle Put this step after a series of steps that are to be repeated for several cycles Gradient Step A Gradient Step i...

Page 48: ...ermal block Right Side Temperature Gradient Enter the desired temperature for the right side of the thermal block Hold for Enter the duration of the Gradient Step units are minutes seconds Convert to...

Page 49: ...aximum starting annealing temperature and the minimum ending annealing temperature Then during the run the software executes the cycling protocol so that the annealing temperature is lowered by an equ...

Page 50: ...nd Hold Time Enter the elongation temperature in the Elongation Temperature field and the duration of elongation in the adjacent Hold For field to be used during touchdown cycling Cycles Enter the num...

Page 51: ...ler 8800 PCR Program File Name PCR Enzyme or Kit Denaturation Annealing Elongation Herculase II Fusion 1kb target pcr Herculase II Fusion DNA Polymerase 95 C for 20 seconds 55 C for 20 seconds 72 C fo...

Page 52: ...ct it Press Run to run the selected program or Open to view the program parameters in the programming screen To edit a preloaded program 1 Press the Programs button on the Home screen 2 In the browser...

Page 53: ...ler 8800 instrument To edit program step parameters 1 Press the Programs button on the Home screen Depending on your user level Guest or registered user the specific user directory or Guest directory...

Page 54: ...the new step See Program Steps in the Commands List on page 43 for details on parameters for a particular command To delete a step from the program press the command in the Program Window to select it...

Page 55: ...eactions in microliters and press OK From the programming screen 1 Press Start 2 In the dialog box enter the volume of the reactions in microliters and press OK To stop or pause a program From the Sta...

Page 56: ...ncubation 1 From the Home screen press Incubation 2 In the Incubation Temperature field enter the temperature of the incubation 3 Specify the duration of the incubation If you want the instrument to h...

Page 57: ...opens the Browse Reports screen listing the report files 2 You can scroll through the list using the arrow buttons at the bottom of the screen or sort the reports by name or date Press the Report Name...

Page 58: ...ng Denaturation times as short as 3 5 seconds may be appropriate Alternatively switch to using capped reaction tubes Increase the reaction volume to 50 l Try switching to the Fast ramp rate If no actu...

Page 59: ...he hot top is disabled Enable the hot top using the instructions in Hot Top Setting on page 32 If the sealed microplate loses sample volume Check that the microplate sealing system is of good quality...

Page 60: ...and unlit Check that the power is on at supply and the power cord is connected Check fuses at rear of instrument above power inlet If screen is lit but shows no information or has missing characters R...

Page 61: ...nd User s Guide Agilent Technologies 5 Maintenance and Updates Maintenance 62 Cleaning 62 Replacing a fuse 62 Software Updates 63 This chapter contains instructions for maintaining the instrument and...

Page 62: ...ent Do not let liquid enter the instrument Turn off and disconnect the instrument from the power supply before you clean Replacing a fuse WARNING Fuse compartments are located on the rear of the instr...

Page 63: ...ator on page 25 for instructions 3 From the Home screen press Settings and then press Software Update 4 Insert the USB memory stick into either of the USB ports on the front of the cycler 5 Press Brow...

Page 64: ...5 Maintenance and Updates Software Updates 64 Agilent SureCycler 8800 Setup and User s Guide...

Page 65: ......

Page 66: ...Agilent Technologies Inc 2011 Version A March 2011 G8800 90000 G8800 90000 www agilent com Agilent Technologies In This Book This document describes how to program and use the Agilent SureCycler 8800...

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