"Transcriptomics & Functional Genomics"

"Update"

Bio-Rads CFX96 and CFX connect Real-time qPCR instruments

In 2011 EUC funded the purchase of a CFX96 and more recently in 2013 added to this capacity with a new real-time PCR instrument CFX connect. These instruments, the Bio-Rad CFX96 and CFX connect are currently housed in the Annex laboratory in Medical Biomics level 2 rm 058. They have advanced capability covering all real-time chemistries including as TaqMan and Sybr Green, they are also capable of HRM and FRET.

All users wishing to use of the instruments including BSc project students must have training before accessing these instruments and are encouraged to contact Dr K Laing. Training will be provided on a personalised adhoc basis along with on-going support as appropriate to the users needs.

FAQs

How do I access the CFX96 or CFX connect and what are the charges

  • No charge will be made for access to these instruments. Electronic access to the room 2.058 on corridor 3a requires card access activation, in which case you need to fill out the access form you can down load it from here. Authorisation can be provided by Dr Ken Laing.
  • Prior training on the use of the instrument is a requirement for access to the instruments and electronic calendars for booking. This can be obtained by contacting Dr Ken Laing klaing@sgul.ac.uk
  • Advanced Booking should be made, noting the time and duration you wish to use the instrument, giving your name and contact details. This can be done on-line using a Microsoft exchange calendar using Outlook or the OWA for each of the instruments.
  • From time to time more information will become available on-line click here

Which machine should I use the CFX96 or CFX connect ?

  • The two instruments are identical in almost all respects, with the exception of the dye channels available to the user. The CFX96 is an instrument that has 5 dye channels that covers all commonly used dyes and can multiplex using all five channels. The CFX connect has three channels but these include the two most frequently used, Sybr/Fam and Vic/Hex. Users adopting these dyes can use the two instruments interchangeably without problems.
  • Each machine is operated using a separate lap top. The data should be saved to a personal folder on the desk top identifying the user by name and can be retrieved using a USB flash drive.

How do I Book on-line using a Microsoft Exchange calendar

  1. With both shared calendars subscription is via invitation. To be added to the calendar, you should first contact Dr Ken Laing at klaing@sgul.ac.uk and arrange training as required and you will then be emailed an exchange calendar invitation.
  2. To install this calendar in the OWA, open the OWA and log in. You will see an email invitation to the calendar in your inbox.
  3. In the Outlook web app (OWA) the email will contain a "+" sign with adjacent text "ADD CALENDAR" this can be found just below the recipient list in the header, click this to add the calendar.
Outlook Web App (OWA)
  1. To add in the calendar using the Microsoft Outlook 2010 email client open this application and log in. Where you will see an email invitation to the calendar in your inbox.
  2. Using Microsoft Outlook the email will have an exchange calendar icon at the top of the email next to the text "open calendar" click this icon to add the calendar.
Outlook 2010
  1. In each case the calendar will appear in the application on the left hand side when you select to display your calendars. A check box should allow you to display or hide the respective calendars as you wish.
  2. Select the check box"CFX96 qPCR booking" and "CFX_connect qPCR booking" calendars to see the current bookings on the two machines. In the OWA these will be displayed overlayed by default, in Outlook you have to select this option.
  3. To add a booking in Microsoft Outlook, click on the time you would like to use the instrument, the tabbed calendar uppermost in the panel is the active calendar into which your booking will be inserted (for example in the image below this is the CFX96). Enter your name and extn, press the carriage return to enter the booking.
  4. Now you may adjust the duration of the booking to the length of your run. To do this put your mouse over the bottom of the coloured block showing the booking you've just made and the cursor will change to a double ended arrow, click and drag to extend or reduce the duration. Your booking is complete. If you need drag and drop the booking to a different time if need be or select and delete the booking.
  1. Please do not book the machine for periods unreasonably longer than necessary, your run protocol will give you an estimate of this duration. If you have to cancel a run delete your booking and release the time for others to use. Please also dispose of your used plates and other rubbish!

What reagents and consumables do the instruments require

The two instruments are compatible with all commonly used chemistries and dyes, plastic consumables can be obtained from any preferred source however, these MUST be a low profile type and can be either both 0.1 and 0.2 ml plates strips and tubes. If tubes or strips are used these should be place within the block so as not to cause undue torsion of the instruments heated lid. See below:

  1. Reagents
    • The open nature of the platform means Regents can be sourced from any supplier (Bio-Rad, Agilent/Strategene, ABI, Thermofisher, PrimerDesign, Solis BioDyne etc), the instrument is compatible with both standard or FAST reagents (some of these suppliers provide discounts and pricing is listed either on Sciene Warehouse others quoatations/pricing are available direct). I and others regularly use these sources.
    • The CFX is an open platform; this means it is compatible with most dyes and reagents. It does not require ROX as a passive reference but if users wish to use ROX this may be done.
    • This means that if users wish to use reagent that were purchased for use with the Mx3000 series they may continue to use those reagents, but may wish upon their next purchase to exclude ROX from their reagent as it is unnecessary.
  2. Plasctics
    • The CFX takes “low profile” tubes/plates these are usually 0.1ml.
    • Ultra-Clear seals, domed and flat caps are all compatible
    • See below

Many manufacturers make compatible plastic ware but these are some example plates/tubes include:

  • Bio-Rad (Very good Discounted pricing is available on the SGUL system)
    • Low profile clear non-skirted PCR plate, natural, 25 plates #MLL9601
    • Multiplate Low-Profile 48-Well Unskirted PCR Plates, 8 x 6 format, natural color, thin-wall polypropylene, pkg of 50 #MLL-4801
    • Full skirted Hard shell PCR-Plate, low-profile transparent or white,#HSP-9xxx
    • Microseal B film Optically clear adhesive Seals pack 100, #MSB1001
    • Low-Profile 0.2 ml 8-Tube Strips Without Caps, natural, 120 strips (960 tubes),#TLS-0801
    • Low-Profile 0.2 ml 8-Tube Strips Without Caps, white, 120 strips (960 tubes),#TLS-0851
    • Optical Flat 8-Cap Strips, for 0.2 ml tubes and plates, ultraclear, 120,#TCS-0803
  • Thermo Scientific (ABgene) available through Fisher (Discount quotation available)
    • Fully skirted low profile plates recommended cut corner H1,#AB-0800, AB-0800/w, AB-2800
    • Non skirted low profile plates alternative cut corner H12, #AB-0700
    • Low profile tube Strips of 8 with ultraclear caps,#AB-1770
    • 50 sheets clear adhesive seal, #AB-1170
    • 120 ultraclear caps, #AB-0866
  • StarLab
    • Skirted Plates for CFX E1403-5200 (clear, low profile)
    • E1403-5209 (same as above, but white)
    • Seals E2796-9795 for qPCR (optically clear) These are "low-tack" seals and as such will not feel sticky at first - as soon as they are applied to the plate using a seal applicator, they give an excellent seal.
  • Eppendorf
    • twin.tec PCR plate low profile skirted #0030 128.672

What Chemistries can I use

  • The CFX96 is compatible with all commonly used chemistries (including TaqMan, Syber Green/Eva Green) and dyes (FAM/Sybr/Eva, VIC/HEX/TET, ROX/Texas Red, Cy5....etc ) and real time PCR applications it is also capable of high resolution melting HRM and FRET
  • The CFX connect is compatible with the most commonly used chemistries (including TaqMan, Syber Green/Eva Green) and dyes (FAM/Sybr/Eva, VIC/HEX/TET, it does not include ROX so mixes that contain ROX should be avoided) and real time PCR applications it is also capable of high resolution melting HRM and FRET.
  • The Bio-Rad CFX96 and CFX connect doe not require or use a passive dye.

What software is available to analyse my data locally

  • We have recently updated the firmware and software and are currently using CFXManager v3.1. This software can be used locally to open your run-files, perform common methodologies for gene expression analysis, allelic discrimination etc and export data to other packages such as biogazelle qBase plus, excel etc. The software is intuitive and easy to use.
  • Users wishing to perform HRM, separate software to import and analyse run-files is available. Contact Ken Laing to obtain a copy of the software and a dongle.

More information generally about Real-time PCR

  • More detailed support and advice for this and other Real-time instruments can be obtained by contacting me Ken Laing klaing@sgul.ac.uk
  • For more general information see my own web pages at http://www.ipc.nxgenomics.org/newsletter.htm and http://www.ipc.nxgenomics.org/
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