1. Performance of Applied Biosystems™ consumables on Bio-Rad CFX™ Real-Time PCR Detection System
February 15, 2017

2. Applied Biosystems™ Consumables on Bio-Rad CFX™
Demonstrate performance of Applied Biosystems consumables on the Bio- Rad CFX (96 & 384 well) instruments.
TaqMan® Fast Advanced Master Mix with TaqMan® Gene Expression Assays
Singleplex vs Duplex Performance
Reproducibility
Dynamic Range
Power Up™ SYBR™ Green Master Mix
Specificity

3. Materials TaqMan® Fast Advanced master mix Assays:
18S Hs _s1 VIC-Primer limited
GPR6 Hs _s1 FAM
PPIA Hs _m1 FAM
YWHAZ Hs _g1 FAM
cDNA: UHR
PowerUp™ SYBR™ Green Master Mix
Targets (all primers run at final concentration of 300nM)
A2M APOA1
ABCC2 ARL1
ADAM10 C11orf30
AOF1 PGK1
cDNA: Liver
All reactions run in quadruplicates
CFX96 – 20ul rxns
CFX384 – 10ul rxns

4. FAMM protocol on Bio-Rad CFX96™ or CFX384™
Protocol run with FAMM is boxed in black. No changes need to be made to run Applied Biosystems™ master mix and assays on the Bio-Rad CFX 96/384 instruments.

5. Singleplex vs Duplex Performance
3 genes + 18S
Average dCq between singleplex & duplex are comparable.
T-Test shows no significant difference between singleplex & duplex
There are no setting for ROX – so data is not normalized to ROX
Only difference in graphs is that one has target labels next to data points, and the other has target names on X axis
GPR PPIA YWHAZ

6. Reproducibility Data shows excellent reproducibility
From dynamic range 18S, PPIA, GAPDH
From singleplex data
18S
PPIA
GPR6
YWHAZ

7. FAMM 6 log dynamic range PPIA 18S
Dynamic range is likely wider, but didn’t run higher concentrations of cDNA

8. FAMM – 18S (VIC Primer limited)

9. FAMM – PPIA

10. FAMM - GAPDH

11. PowerUp™ SYBR™ Green Master Mix
95 deg / 2min
95 deg/15sec x40 cycles
60 deg/ 1 min
Dissoc curve

12. Dynamic Range - PowerUp™ SYBR™ Green Master Mix
Excellent reproducibility
3 targets
2 targets (PGK1& ARL1) – 4 logs
1 target –(ABCC2) - 2 logs

13. Dynamic Range - PowerUp™ SYBR™ Green Master Mix
Same data as previous slide, with
ABCC2 data removed

14. PowerUp™ SYBR™ Green Master Mix - PGK1

15. PowerUp™ SYBR™ Green Master Mix – ARL1

16. PowerUp™ SYBR™ Green Master Mix – ABCC2

17. PowerUp™ SYBR™ Green Master Mix -6 targets - Reproducibility
A2M, AOF1, ARL1
ADAM10, APOA1, C11orf30

18. PowerUp™ SYBR Green Master Mix – Excellent Specificity – Dissociation Data
APOA1
ARL1
C11orf30
A2M
ADAM10
AOF1
No non-specific amplification or primer dimer

19. PowerUp™ SYBR™ Green Master Mix – Excellent Reproducibility

20. PowerUp™ SYBR™ Green Master Mix – Excellent Reproducibility
All data from all 6 genes plus 3 dil curves

21. Conclusions and Next Steps
Applied Biosystems™ consumables perform well on the Bio-Rad CFX™ (96 & 384 well platforms)
TaqMan® Gene Expression Assays run with TaqMan® Fast Advanced Master Mix shows excellent reproducibility, dynamic range and equivalent results in singleplex vs duplex.
PowerUp™ SYBR™ Green Master Mix shows excellent reproducibility and sensitivity.
To learn more about Applied Biosystems qPCR master mixes or to request a free sample, please visit
To download the ‘Design and Optimization of SYBR Green Assays’ and/or ‘Protocol for Comparing SYBR Green Master Mixes’ please visit
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PowerUp
PGK1 – 4 logs
ARL1 – 4 logs
ABCC2 – 2 logs
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