PCR Thermal Cyclers Support - Troubleshooting

PCR is not perfect. Each step of the cycle should be optimized for each template and primer pair combination. The most important considerations for any PCR reaction are:

• PCR enzymes—enzyme selection can affect the yield and specificity of PCR products. Get the basics on how to select the right PCR enzyme for your application.
• Primer design—optimize your primer design using our custom oligo design tool.
• Magnesium concentration—the correct magnesium concentration needs to be determined empirically for each primer and template combination you have.
• Annealing temperature—the appropriate annealing temperature is based on the calculated Tm of your primers; watch the below video on better than gradient to optimize your annealing temperature.

The heated thermal cycler lid is designed to make sure that optimized pressure is applied on the microplate for an efficient PCR reaction. When using PCR tubes, excess pressure on the tubes, from the heated lid can cause deformation of the tubes. To avoid this, we recommend using the Tray/Retainer set, as shown below:

• Cat. No. 4381850, MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the Veriti™ Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.
• Cat. No. 403081, MicroAmp™ 96-Well Tray/Retainer Set – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the 7000 System, 2720 Thermal Cycler, and GeneAmp™ PCR System 9700.
• Cat. No. 4379983, MicroAmp™ 96-Well Tray for VeriFlex™ Blocks – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes with Caps, 0.2 mL), with the Veriti™ Thermal Cycler, Veriti™ Fast Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.

Please refer to this guide to restore calibration values.

The heated thermal cycler lid is designed to make sure that optimized pressure is applied on the microplate for an efficient PCR reaction. When using PCR tubes, excess pressure on the tubes, from the heated lid can cause deformation of the tubes. To avoid this, we recommend using the Tray/Retainer set, as shown below:

• Cat. No. 4381850, MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the Veriti™ Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.
• Cat. No. 403081, MicroAmp™ 96-Well Tray/Retainer Set – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the 7000 System, 2720 Thermal Cycler, and GeneAmp™ PCR System 9700.
• Cat. No. 4379983, MicroAmp™ 96-Well Tray for VeriFlex™ Blocks – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes with Caps, 0.2 mL), with the Veriti™ Thermal Cycler, Veriti™ Fast Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.

First, make sure you are running the most up to date firmware. Newer firmware versions have fixed many wireless and mobile app issues. Firmware updates can be found here.

To set up the wireless connection in the ProFlex™ PCR machine, please follow the steps outlined below:

1. Power on the ProFlex™ Instrument, and in the Home screen, touch Settings.
2. In the Settings screen, touch Device Settings.
3. In the Device Settings screen, touch Network Connection. In the Network Connection Type screen, touch Wireless.

   The Search Wireless Network screen will come up and the wireless symbol will appear active. Touch Cancel to exit the screen.
   Note: During initial setup, if you selected the Wired option in the Network Connection Type screen, you will be required to enter your IP address if you selected the Static IP wired option. If you selected the Dynamic IP wired option, the IP address is automatically populated.

4. Once a wireless connection has been detected, a list of the available networks will be displayed in the Choose Network screen. Touch the network name of your choice or touch Join Other Network.
5. In the Find and Join a Network screen, enter the name and security type of the network.

   Note: When you touch in the Network Name field, a keypad will come up to facilitate making the entry.

6. From the Security type drop-down menu, touch to select the security type and enter the relevant information in the screen.

   Note: Contact your IT Systems Administrator for information on security type.
   You can select from the following options:

   • WEP
   • WPA Personal
   • WPA2 Personal
   • WPA Enterprise
   • WPA2 Enterprise

7. Touch Join to continue or Cancel to exit from the Find and Join a Network screen.
8. Depending on the security type you have selected, enter the appropriate passwords and touch Join.

   If you entered the correct information, the Network Connection Complete screen will appear. Touch OK to continue. If you entered incorrect information, the Network Connection Failed screen will come up and you will be required to go back to security type screen to resolve the issue. Touch OK to continue.

1. Ensure that the instrument is Cloud-enabled.

Any ProFlex PCR System can be connected to Thermo Fisher Connect as long as it is using firmware version 1.2 or higher. You can check your current firmware version by:

 a) Going to the Settings menu.

 b) Selecting “About Instrument”.

2. Create a Thermo Fisher Connect account

 a) From your Internet browser, go to thermofisher.com/connect

 b) If you already have an account with Thermo Fisher Scientific, log in using the same name and password.

 c) If you do not already have an account, follow the prompts to create one.

 d) Once you have logged in to thermofisher.com/connect, an email will automatically be sent to you  to verify your email address.

 e) Open your email and click on the link to visit the Connect page to verify your account.

 f) If you have done this successfully, you will receive a confirmation email telling you that you have connected to Thermo Fisher Connect.

3. Connect your ProFlex PCR machine to the internet using a wireless or wired connection.

Note: Please refer to Chapter 2 of the ProFlex PCR System User Guide for instructions on setting up of a wireless or wired connection.

4. Connect the instrument to Thermo Fisher Cloud

 a) Touch Sign In.

 b) Select Administrator when asked to choose screen name.

 c) Log in. (The default password for the Administrator account is “password”.)

 d) You will then be redirected back to the home screen. Go to Settings.

 e) Select Remote Service.

 f) Turn “on” Remote Service.

 g) Touch My Profile on the home screen.

 h) Touch the Cloud tab and enter your Thermo Fisher Connect account login and password.

 i) Select Link Account. Upon successful authentication, the Administrator is linked to Thermo Fisher Connect.

 - Subsequently, any user can log in and link the instrument to their Thermo Fisher Connect account by following steps 3-9 above.

Note: The administrator account must be activated first to connect the instrument to the Cloud. Afterward, multiple users can create unique user profiles using their own login information. Each unique profile must have a unique Thermo Fisher Connect account.

5. Set mobile connectivity in a mobile device

 a) Download and install the "Instrument Connect Mobile Application" on your mobile device.

 - For an iPad™ or iPhone™ mobile device, download the application from the App Store™ by searching for “Instrument Connect”.

 - For Android™ devices, download the application from Google™ Play by searching for “Instrument Connect”.

 b) Log in to the Instrument Connect mobile application using your Thermo Fisher Connect login and password.

 - Once logged in, any Cloud-enabled instrument that has been linked to that account can be monitored.

The heated thermal cycler lid is designed to make sure that optimized pressure is applied on the microplate for an efficient PCR reaction. When using PCR tubes, excess pressure on the tubes, from the heated lid can cause deformation of the tubes. To avoid this, we recommend using the Tray/Retainer set, as shown below:

• Cat. No. 4381850, MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the Veriti™ Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.
• Cat. No. 403081, MicroAmp™ 96-Well Tray/Retainer Set – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the 7000 System, 2720 Thermal Cycler, and GeneAmp™ PCR System 9700.
• Cat. No. 4379983, MicroAmp™ 96-Well Tray for VeriFlex™ Blocks – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes with Caps, 0.2 mL), with the Veriti™ Thermal Cycler, Veriti™ Fast Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.

Make sure you have the most up-to-date firmware version, as this has fixed many screen-freezing issues. The latest firmware can be downloaded from here. If you cannot get access to the main menu, try turning off the machine, letting it sit for 1 minute, and restarting it without any block attached.

If the test fails:

1. Verify that the:
   • Calibration of the TVK is up-to-date.
   • Probe tips are not cracked or damaged.
2. Dispense 2 to 3 drops of mineral oil onto a cotton swab. Use the swab to apply a thin film of oil to the cone surface of each probe to improve heat transfer.
3. Repeat the steps in “Before You Run 96-Well Instrument Tests” from the Multiprobe Temperature Verification Kit User Guide.
4. Verify that the:
   • Probes are seated securely in the appropriate test wells.
   • Cables are connected securely to the ports.
   • Ribbon cable lies flat under the thermal cutout and is not crimped.
   • Heated cover is closed and locked down.
5. Repeat the steps in “Run the Test” from the Multiprobe Temperature Verification Kit User Guide.

Please note, if the test fails again, contact Life Technologies at instrumentservices@lifetech.com.

The heated thermal cycler lid is designed to make sure that optimized pressure is applied on the microplate for an efficient PCR reaction. When using PCR tubes, excess pressure on the tubes, from the heated lid can cause deformation of the tubes. To avoid this, we recommend using the Tray/Retainer set, as shown below:

• Cat. No. 4381850, MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the Veriti™ Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.
• Cat. No. 403081, MicroAmp™ 96-Well Tray/Retainer Set – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the 7000 System, 2720 Thermal Cycler, and GeneAmp™ PCR System 9700.
• Cat. No. 4379983, MicroAmp™ 96-Well Tray for VeriFlex™ Blocks – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes with Caps, 0.2 mL), with the Veriti™ Thermal Cycler, Veriti™ Fast Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.

A guide to replacing lost calibration files for the GeneAmp® PCR System 9700can be found here.

The heated thermal cycler lid is designed to make sure that optimized pressure is applied on the microplate for an efficient PCR reaction. When using PCR tubes, excess pressure on the tubes, from the heated lid can cause deformation of the tubes. To avoid this, we recommend using the Tray/Retainer set, as shown below:

• Cat. No. 4381850, MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the Veriti™ Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.
• Cat. No. 403081, MicroAmp™ 96-Well Tray/Retainer Set – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes without Caps, 0.2 mL), with the 7000 System, 2720 Thermal Cycler, and GeneAmp™ PCR System 9700.
• Cat. No. 4379983, MicroAmp™ 96-Well Tray for VeriFlex™ Blocks – This Tray is for use with MicroAmp® Strip Tubes (or MicroAmp® Reaction Tubes with Caps, 0.2 mL), with the Veriti™ Thermal Cycler, Veriti™ Fast Thermal Cycler, SimpliAmp™ Thermal Cycler, and Proflex™ PCR System.