qPCR plate preparation – Robotic method

Rev 1.0 (29/06/2017)

Reagents and Materials included in ADNucleis kit :

  • The RT-PCR kits are distributed in bulks of 48 reactions under the form of a 2X concentrated premix.
  • Premix: The dilution of the 2x premix is managed by the robot using ELU buffer.  The 2X concentrated PCR premix contains all the ingredients necessary for the detection of the target (s) of the PRRS virus (primers, dNTPs…) and the IPC primer/probes, except enzymes.
  • Taq (Taq polymerase Nucleis used to amplify the DNA)
  • IPC / IAC _ARN  (RNA inhibition control)
  • RT (Reverse transcriptase Nucleis used to transcribe RNA into cDNA)
  • Elution buffer ELU
  • RNA positive control  (200µL/ tube of positive control is provided for each vial of 48 reactions)

Reagents and Materials not included in ADNucleis kit :

  • Recommended consumables
    • 96 well PCR Plate PCR (Be careful to use a plate suitable for both the Inheco device and your qPCR thermocycler)
    • QPCR Sealing film (e.g. ThermalSeal RT2RR)

  • Upon reception, ADNucleis amplification kits must be stored at -20°C until use.
  • DO NOT freeze back or reuse a reconstituted PCR 1x mix.
  • The reverse transcription step to generate cDNA from RNA is performed separately from the PCR. The RT step is carried out first by adding the RT enzyme in the premix manually. When the RT step is done, the Taq polymerase Nucleis is added t the mix by the robot in each well.
  • ADNucleis recommends to avoid freeze/thaw cycles (Not >3) of the amplification kits.
  • Vials of PCR premix  can be aliquoted to avoid excessive thawing and freezing when premix are thawed for the first time (8, 16 reactions). Aliquoted premix must be kept at -20°C.
  • If the component of the kits are not aliquoted, please write on the original  vials the number of thaws and volumes pipetted.

Example of annoted vials

Numbers indicate  the number of reactions  used  16, 12 and 4 puits; indicating 3 different thaws.

PROCEDURE

Generate a PCR layout and the worklist using ADN_SOFT

 

  • Choose the desired target (s) (SELECT_TARGET), and write an “x” in each well to be analyzed with the corresponding target.
    • For detection mode, the genomic quantification can be left blank. The bottom of the table shows the fluorescent channel used for each target.
  • Create the layout code for the robot by clicking “Create PCR plate(s)”
  • Fill the cell plate number (If there are several PCR plates to run all the samples extracted, the PCR plate can be generate successively by indicate the plate number). When 94 samples are tested with one target, only one PCR plate will be created (94 + 1 negative control and 1 positive control = 96 used wells). Fill the cell with “1”.
  • Generate the worklist by clicking “Generate Code”

Amplification mix reconstitution

  • Work with ice or cold bloc. Inheco blocks can be used; To use the inheco device manually: Click “Temp” if the temperature is already set to 4°C. Otherwise, click “set”, then “4” and “0”, then click on the left top button when the temperature is written “004.0°C”. Then click “Temp”. The button “Set” should indicate 4°C.
  • If extracted samples have been stored at 4°C or frozen before the purification step, the deepwell containing samples should warm on the thermoshaker “Inheco DW 96 Ext” of the robot set to 4.0°C and thaw for 10 min at this temperature.
  • Thaw HQS_xxx_PRE-mix premix vials, IPC vials and the RT Nucleis vials using the 24 well Inheco block of the robot. Put the vials in each slot according to the map given by the ADN_soft. Put an empty vials to reconstitute the amplification mix.
  • The mix reconstitution is performed according to the table below
 Component Volume per well (µl) Volume per Y well (µl)
HQS_xxx_pre-mix (with IPC) 18 18 x Y
RT Nucleis 1 1x Y
IPC 3 3 x Y
reconstituted Mix(µl) 22 22 x Y

NB: ADN_SOFT will show the different volumes for the mix reconstitution. The software  takes into account the number of reactions and loss of reagent due to pipetting. Volumes of reagents provided by ADNUCLEIS in each kits take into account this loss of reagents due to pipetting. The 48 reactions kits contain enough volume for 58 reactions).

BE CAREFUL :

  1. As well as for PVG, NO VOLUTE should remain after homogenization. Pipet 10 times in order to homogenize correctly Premix with RT and then with IPC.
  2. No bubbles should be seen inside tubes (reconstituted mastermix , positive rna, Taq)

Starting the robot and QPCR

  • Put a  1.5 ml vial into the slot indicated by ADN_SOFT, add the volume of Taq given by ADN_Soft.
  • Add 10 ml of ELU buffer and 20 ml of D1 buffer into the indicated container.
  • Check if the liquid system container contains enough volume of 2% D3. Fill it if necessary according to the volume indicated by ADN_SOFT. Be careful when filling the container. Add the water first, then the adequate volume of concentrated D3 to avoid foaming.
  • Empty the waste container
  • Verify the reconstituted mix, Taq, RNA positive control vials are correctly placed according to the 24 well Inheco device map on ADN_SOFT.
  • Put a new PCR plate on the PCR Inheco device.
  • Click “flush” in Gemini

  • Then, click “initialize”

  • To run the program click file and choose the program “ADNucleis_qPCR_plate_prep”, then click “Play”
  • Follow the program
  • At the end the run (around 30 min + 1 min per sample), seal the PCR plate and put it immediately in the QPCR apparatus and launch the QPCR program. If you do not run the QPCR immediatly, then freeze immediatly the PCR plate at -20°C until use.
Posted in Technical notice.