Realtime Quantitative PCR

Available devices

Light Cycler 480 – Roche

  • Blocks for 96 and 384 well microplates
  • SYBRGreen detection, Taqman
  • Genotyping and HRM

Pipeting Robot Biomek3000

  • Computer station for the control and the programming  of the robot
  • Precise pipetting from 1 μL – 200 μL for single and 8-channels configurations

Access to the consumables

SYBR Green

You can buy 2 ml tubes of SYBR Green mix in the store (reference 10050-Roche and 10079-Eurogentec).

 384 well microplates and optical films

The 384 well microplates and films are situated next to the LightCycler in the lab.

Tips for the Biomek robot

You wil find the P20, P50 and P200 tips under the LightCycler.

Access to the devices and reservation

  •  If you don’t know how to use the devices, please ask the platform’s members or other confirmed users,
  • Use the utilization procedures for the LightCycler480 and the (available in the lab, don’t print them) Biomek3000
  • Do not install software on the computers,
  • Reservations of the LC480 and the Biomek 3000 are mandatory via the IBMP intranet. Reservations are made of 1 hour slots corresponding to a standrad run on the LC480.
  • Please respect your reservation in order not to penalize the following users,
  • Find the procedures and protocoles for instruments use hereafter. Please don’t print them as they are available in the lab.

Rules of conduct LyghtCycler qPCR

  •  On the LightCycler, save your run in the folder assigned to your team, where you can create a subfolder with your name : Roots> QPCR 406> Experiments> QPCR 2020> Team Folder> User Folder,
  • Retrieve your .ixo files (an annual backup will be performed on the IBMP server),
  • The computer should only be used to control the instrument and not to analyze your results. To analyze your results, another computer is available on the platform,
  • The lab and instruments have to remain clean after each run : throw your plates away, don’t let any plate in the instrument or on the benchtop,
  • Turn the LC480 and the Biomek3000 off  if no reservation has been scheduled after yours,
  • Report any breakdown or technical failure to the platform members as soon as possible.

Activities

Plate and experiment preparation with the Biomek3000

Protocol Biomek3000 use

Protocol plate preparation for Lightcycler and LightCycler480 use.

Find some reference genes

From a collection of genes; search for the 2 or 3 more stable genes for an organism, for a given tissue or under given experimental conditions,

Softwares used: Genorm , NormFinder , GeneX.

The plateform offers 3 sets to normalize for the following organisms:

– Arabidopsis thaliana

– Nicotiana tobaccum

– Solanum tuberosum

Here is a list of the primers that are available at the platform.

Primers and probes design

design of primers with the best efficiency and efficacity for a given gene,

Softwares used:  LighCycler primer Design, LinReg.

Protocol for primer design with LightCycler primer Design here.

Protocol for efficiency calculation with LinReg here

Analyzes

Relative quantification (ΔΔct and  Δct method) and  absolute quantification analyzes,

Statistical analyzes.

Technical support and softwares

Technical support for the experiment realization and configuration, technical support for the cDNA synthesis.

CNRS training

See Malek if you are interested in the training session