Evaluate the Performance of Real-time PCR Instruments by Using EzMate 401 Automated Pipetting System

Benefits

 

  • EzMate Automated Pipetting System provides μl-level liquid transfers make it suitable to for the performance evaluation of real-time PCR instrument
  • Improve the reproducibility of evaluation using EzMate Automated Pipetting System

 

Introduction

 

The highly accurate and precise EzMate 401 Automated Pipetting System is specialized in handling low-volume pipetting tasks, such as special qPCR experiments, which can be used to evaluate the realtime PCR instrument’s performance. In this application note, we use EzMate 401 to prepare a special qPCR experiment to evaluate the well-to-well variation of a real-time PCR instrument

 

Material

 

Equipment:

 

  • EzMate 401 with APM50-1
  • Roche LightCycler® 480 Real-Time PCR System

 

Reagent:

 

Finnzymes DyNAmo SYBR Green qPCR Kit, # F-415-L

 

Template:

 

101bp synthetic DNA with following sequence,

1X working Conc. 1pM:

AAC TTG GCT TTA ATG GAC CTC CAA TTT TGA GTG TGC ACA AGC TAT AGA ACA

CCA CGT AAG ACA TAA AAC GGC CAC ATA TGG TGC CAT GTA AGG ATG AAT GT

 

Primers:

 

Forward: AAC TTG GCT TTA ATG GAC CTC CA, working Conc. 300nM

Reverse: ACA TTC ATC CTT ACA TGG CAC CA, working Conc. 300nM

 

Consumables:

 

  • Roche LightCycler® 480 Multiwell Plates 384, # 047729749001
  • Axygen 50ul Robotic Tip, w/o filter, Non-Sterile, # FX-50-R

 

Methods

 

Prepare the pre-mix solution (listed in the table 1. below) in a 1.5 ml tube, and invert the tube several times to mix the content thoroughly (do not vortex the tube).

 

 Table1

                                                          Table1

 

Use EzMate 401 to dispense 20 μl/well of the premix solution into each well in columns 1, 12 and  24  of  a  clean  384-well  qPCR  plate  (blue frames in figure 1. below).

 

 

Use EzMate 401 to dispense 20 μl/well of the premix solution into each well in columns 1, 12 and  24  of  a  clean  384-well  qPCR  plate

                                                                 Figure1

 

After dispensing, seal the plate, gently tap its side and then centrifuge it to remove any bubbles in the well. Run the qPCR experiment under the following cycling conditions: (below, table 2.)

Run the qPCR experiment under the following cycling conditions

                                                                                          Table2

 

                                                                        Result

Cp mean = 19.47316854

Cp SD = 0.031756082

Acceptable criteria: SD of Cp should be ≤ 0.5.

 

The test result indicates that the tested instrument performs well. The variations between wells are very small.

 

Conclusion

 

The high accuracy and precision of the EzMate automated pipetting system in μl-level liquid transfers make it a suitable  tool  to prepare  special  qPCR  experiments  for  the performance  evaluation  of  any  real-time  PCR instrument. By using the synthetic oligonucleotide as the target template, users can eliminate potential problems caused by the uncertainty of the natural DNA sample, and improve the reproducibility of the evaluation.

 

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PCR/qPCR Set Up Related Applications

 


 

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