OneDip™ QPCR reproducibility

 

 

 

 

Rob™ OneDip™ QPCR set-up reproducibility for 2μl template


OneDip™ mode aspirate 3 X “template volume” and dispense template in three target wells.
OneDip™ mode saves 65% of tip costs and improves speed for a 96-plate with triplicates by 50%.

The template is a single stranded DNA fragment containing the beta-actin amplicon sequence. Primers are specific for mouse beta-actin.

Results:

 

Based on single template
CqAverage: 21.25
Standard deviation (Cq): 0.19
Cq
Max – CqMin: 0.82
  Based on average Cq value of triplicate
CqAverage: 21.25
Standard deviation (Cq): 0.075
Cq
Max – CqMin: 0.23


Run conditions:
QPCR: HotStart activation, 2 min at 95℃ followed by 40 cycles with 10 s at 95℃ and 10 s at 60℃. Run time: 44 min.
Set-up: 18 μl master mix was dispensed using multi dispense followed by 2 μl template using OneDip™ technology (multi dispense) from one tube. Run time: 4 min 57s.

Reagents:
qPCRBIO SyGreen Mix from PCRBiosystems was selected because it is the fastest and most sensitive kit available on the market. It combines the latest polymerase and buffer technology with a proprietary intercalating dye that doesn’t inhibit PCR.

QPCR instrument:
Mic QPCR-instrument from Bio Molecular Systems is the most precise QPCR instrument available today. Virtually no temperature differences between samples during the run combined with the magnetic induction heating ensures highest possible quality on data.
Reproducibility of Mic is within Cq 0.20.

Conclusion:
OneDip™ mode is useable from 2 μl template volume for detection and concentration calculations. The variance between average triplicate values is less than for individual templates. Larger template volumes will substantially improve precision. Lower volumes are not recommended.
We recommend to use single dispense for accurate expression analysis and concentration determinations for low template volumes.

 

 

AlphaHelix Molecular Diagnostics (Publ.) AB

HEAD OFFICE

Västra Finnbodavägen 2
SE-131 30 Nacka Stockholm
Sweden
+46 (0)862 67 600
info@alphahelix.com

AlphaHelix Technologies AB



Penarp 1150
SE-264 91 Klippan
Sweden
+46 (0)862 67 600
sales@alphahelix.com
© 2017 AlphaHelix Technologies AB
Information om cookies