QPCR reproducibility
Rob™ QPCR set-up reproducibility for 1μl template
The template is a single stranded DNA fragment containing the beta-actin amplicon sequence. Primers are specific for mouse beta-actin.
Result:
CqAverage: 21.35
Standard deviation (Cq): 0.08
CqMax – CqMin: 0.30
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: 45 min.
Set-up: 18 μl master mix was dispensed using multi dispense followed by 1 μl template using single dispense from one tube. Run time: 8 min 14s.
Reagents:
qPCRBIO SyGreen Mix from PCRBiosystems was selected because it is the fastest and most sensitive kit available on the market. It combines 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:
The precise liquid handling performance of Rob™ allowS precise QPCR analysis for template volumes down to 1 μl. The reproducibility data on 1 μl is better than the reproducibility of 96/384 plate QPCR instruments.
Speed
Rob™ has two modes for doing QPCR set-up. Traditional mode uses one tip for every sample replicate and OneDip™ mode uses the same tip for all replicates doing serial dispensing. The example below is based on a QPCR set-up with three replicates and pre-made master mix.
One tip per replicate
96-plate: 27 minutes (18 µl mix + 2 µl sample)
384-plate: 95 minutes (5 µl mix + 2 µl sample)
OneDip™
96-plate: 13 minutes (18 µl mix + 2 µl sample)
384-plate: 45 minutes (5 µl mix + 2 µl sample)
Rob™ handles a 384 set-up with acceptable speed but with low investment cost.
Accuracy & Precision
Accuracy and precision data obtained under optimized conditions may not be possible to reproduce in your application. The data below were obtained under normal QPCR set-up conditions. On the other hand, high precision data indicates that the liquid handling is stable. Market leading precision can easily be reproduced at your lab. High precision and accuracy also grants that dispense performance is very robust. Changes in viscosity will not have the same impact on dispensed volume seen in competing systems. So the dispense data below is what you can expect from Rob™ in reality.
50 ul pipette tip with filter
| Volume (µl) |
Mean (µl) |
Precision (C.V.) |
| 1 |
1.00 |
3.78 |
| 2 |
1.99 |
1.83 |
| 5 |
5.00 |
0.76 |
| 10 |
10.02 |
0.38 |
Ten single dispenses in ten PCR tubes (0.2 ml).
50 ul pipette tip with filter, OneDip™
| Volume (µl) |
Mean (µl) |
Precision (C.V.) |
| 2 |
2.03 |
1.93 |
| 5 |
5.05 |
1.67 |
Three series dispenses in 3 x3 PCR tubes. Tubes were pre-filled with 15 µl master mix to mimic a QPCR set-up.
Liquid level detection (LLD)
It is essential to have in-process LLD and tip detection to verify processed protocols. There are two methods used to measure the liquid level heights. Either conductive or pressure based systems are used. The advantages of pressure based LLD are that all liquids can be detected, pipette tip cost is reduced to almost half and the system isn’t sensitive for static electricity. Conductive measurements require expensive conductive tips, is sensitive to static electricity but measures faster.
Rob™ uses a further developed pressure based LLD to improve detection speed. The LLD system provides with better precision and sensitivity than needed.
Detection limit: Detects as low 3 µl in a 200 µl PCR tube.
Detection precision: ± 0.3 µl in a PCR tube with 20 µl volume.