Optimized concentration of Primer Stabilizer
 
(1) Confirmation of real-time PCR Reaction
We changed concentration of Primer Stabilizer to 0 - 2.5mM and performed real-time PCR with it by SYBR Method and Hydrolysis Probe Method, then compared the results of the two Methods.
 
< Materials · Method >
Reaction sample: cDNA(human WT1, human bcr/abl)
Instrument: LightCycler®480
Enzymes: LightCycler®480 SYBR Greenâ… Master, and LightCycler® Probe Master
 
< Result >
Human WT1Human bcr/abl
SYBR DetectionHydrolysis Probe Detection
Primer Stabilizer ConcentrationCP ValueCP Value
0mM22.8523.05
0.5mM22.8822.77
1mM23.3622.58
2.5mM25.4222.91
 
Example of detected wave sharp, Human WT1(SYBR)
 
< Discussion >
Optimized concentration of Primer Stabilizer is 0.5 - 1.0mM in final concentration of reaction system and it did not inhibit real-time PCR. Primers added with this concentration range of Primer Stabilizer are usable only by diluting it into working solution.
 
(2) Confirmation of High Resolution Melting(HRM)
< Purpose >
We evaluated influence of Primer Stabilizer on Melting Curve by HR-1 Control Kit.
< Material · Instruments>
Primer: Primer of HR-1 Control Kit(Human β3 adrenergic receptor)
Master Mix: LightCycler®480 High Resolution Melting Master
Instrument: LightCycler®480
Sample: GenomeDNA10ng(C/T Hetero, DNA extracted from saliba)
< Method >
Reaction Components :Concentration of Primer 0.5µM, template 10ng, others are according to Standard Protocol of Master Mix Kit.
Concentration of Stabilizer: We added Stabilizer so that final concentration of reaction system should become 0, 0.25, 1.0, 2.0, and 3.0mM.
Temperature Conditions:Annealing temperature 60℃, extension reaction time 5 sec., others are according to Standard Protocol of Master Mix Kit.
< Result >
here was no influence on Melting Curve at the final concentration of Primer Stabilizer at 0 - 3.0mM
< Discussion >
From this result, there was no influence on Melting Curve at the optimized concentration of Primer Stabilizer at 0.5 - 1.0mM obtained from the Experiment(1). No inhibition was observed by addition of Primer Stabilizer in qualitative·SNP Analysis in PCR reactions.