Biosharp BL698A Fluorescent Quantitative PCR Kit (5X1mL)
Manufacturer introduction:
Biosharp BL698A Fluorescent Quantitative PCR Kit (SYBR Green qPCR Mix)
Brand:biosharp
Item number:BL698A
Specifications:5X1mL
This product is made usingSYBR Green IChimeric fluorescence methodReal Time PCRSpecial reagents for hot startTaq DNAPolymerasedNTP、SYBR Green IReady to use once the reagents are pre mixed2x Mix, can targetcDNAPerform rapid and highly specific quantitative detection. This product is chemically modifiedHot Start Taq DNAPolymerase, in50 ℃The following is inactive, only95 ℃Under certain conditions, the enzyme's activity can only be fully restored after heating, ZMinimize the production of non-specific amplification products and improve fluorescence quantificationPCRThe accuracy of the reaction. The unique reaction buffer of this kit can achieve good amplification results in a wide range, higher detection sensitivity, and stronger signal.
Product components:
product number |
product name |
specifications |
1 |
2×SYBR Green qPCR Mix |
5x1ml |
2 |
50×ROX Reference Dye |
1ml |
storage condition:
After receiving this product, please place it immediately-20℃Store in the dark, valid for one year,2~8℃Storage under conditions6Month,Avoid repeated freeze-thaw cycles.
instructions:
1、 PreparationReal-Time PCRReaction system:
1Collect all reagents2×SYBR Green qPCR Mix,ROX Reference DyeTemplates, primers, andRNase-Free ddH2ODissolve and thoroughly mix at room temperature,Avoid generating bubbles.After brief centrifugation, placed on icePrepare the reaction solution according to the table below.
Refer to the table below to prepare the reaction system:
component |
20ulsystem |
final concentration |
SYBR Green qPCR Mix |
10μl |
1× |
Primer F (10 μM)* |
0.4~0.8 μl |
0.2~0.4 uM* |
Primer R (10 μM)* |
0.4~0.8 μl |
0.2~0.4 uM* |
50 x ROX Reference Dye** |
- |
- |
cDNAtemplate |
- |
-- |
RNase-Free ddH2O |
to20 μl |
- |
* Generally speaking, the final concentration of primers in the reaction system is0.2 uMA better amplification effect can be obtained. When the reaction performance is poor, it can be achieved at the final concentration0.2~0.4 uMAdjust the primer concentration within the range.
** Z adaptability of several common instrumentsROX Reference DyeThe concentration is shown in the table below:
Instrument model |
ppm |
ABI PRISM 7000/7300/7700/7900HT/StepOne/StepOnePlusclass |
5× |
ABI 7500/7500 Fast、QuantStudio ® 3/5、QuantStudio 6/7 Flex、ViiA 7、Stratagene Mx3000P/Mx3005PandMx4000class |
1× |
Roche、Bio-Rad、Eppendorfclass |
No need to add |
2Cover or seal the reaction tube/PCRPlate, gently mix well. Briefly centrifuge to ensure that all components are in the tube/Bottom of the board.
2、 Carry outReal-Time PCRreaction
1、Suggest adopting a two-step approachPCRThe reaction program carries out the reaction.
Two-step reaction program:
step |
temperature |
duration |
Number of cycles |
Pre denaturation |
95℃ |
5min |
1 |
sex change |
95℃ |
10sec |
40 |
Annealing extension data acquisition |
60℃ |
30sec |
40 |
melting curve analysis |
According to the recommended program settings for the instrument |
equalFactors such as low template quantity can lead to poor amplification effect, and a three-step program can be used for this purposePCRReaction.
Three-step reaction program:
step |
temperature |
duration |
Number of cycles |
Pre denaturation |
95℃ |
5min |
1 |
sex change |
95℃ |
10sec |
40 |
annealing |
50-60℃ |
30sec |
40 |
Extended data collection |
72℃ |
30sec |
40 |
melting curve analysis |
According to the recommended program settings for the instrument |
2、Machine detection: Place the reaction system in fluorescence quantificationPCRIn the instrument, run the program to collect data and analyze the results.
Biosharp BL698A Fluorescent Quantitative PCR Kit (5X1mL)