Product Description
The 2x Gene-Foci® PCR master mix is a pre-mixed 2x solution of Taq DNA polymerase, PCR reaction buffer, dNTPs, MgCl2, enzyme stabilizer, PCR enhancers and SYBR® Green fluorescein. The master mix contains all the necessary components for qPCR amplification except template DNA and primers. This PCR master mix has been optimized for high-efficiency qPCR amplification, and is ideal for quantifying cDNA and genomic DNA.
Features:
Super active and thermal-stable DNA polymerase in the master mix guarantees stable and robust amplification of target sequence.
PCR enhancers and enzyme stabilizers in the master mix make the qPCR reaction more reproducible.
Ultra sensitive, can detect trace amount of genomic DNA or cDNA.
Affordable, significantly cheaper than similar products from competitors.
Storage :
For long term storage, keep at -80°C or -20°C. Shield against light. For short term storage, thaw the master mix and store at 4°C. The master mix is stable at 4°C for at least one month. Avoid repeated freeze and thaws.
Fig. 1. The cDNA synthesized using Gene-Foci® High Efficiency Reverse transcription kit was diluted 1:200, 1:500, 1:2000 and 1:5000, then 18S rRNA expression level was examined with Gene-Foci® SYBR® Green qCR Master Mix.
qPCR Reaction Setup
To setup a 25µl qPCR reaction, add the following components into a DNase-Free qPCR plate or tube:
|
Components |
Volume |
Final Concentration |
|
2X Fast PCR Master Mix |
12.5 µl |
1X |
|
Forward Primer (1 μM) |
1~7.5 µl |
50~300 nM[1] |
|
Reverse Primer (1 μM) |
1~7.5 µl |
50~300 nM[1] |
|
DNA Template |
1~5 µl |
0.1~10 ng/μl |
|
ddH2O |
to 25 µl |
[1] A primer titration from 50 nM to 300 nM final concentration should be performed to determine the most amplification-efficient concentration.
qPCR Thermal Cycling Program
Set up the program qPCR on a qPCR machine as follows:
Option I: Two-step PCR
|
Cycles |
Temperature |
Time |
|
| (1) Pre-denature |
1 |
95°C |
3 minutes |
| (2) Thermal cycling |
40 |
95°C |
15 seconds |
|
55~60°C(a) |
30~60 seconds |
Option II: Three-step PCR
|
Cycles |
Temperature |
Time |
|
| (1) Pre-denature |
1 |
95°C |
3 minutes |
| (2) Thermal cycling |
40 |
95°C |
15 seconds |
|
55~60°C(a) |
20 seconds |
||
|
72°C |
30 seconds |
(a) Temperature required for annealing/extension step may need adjustment for different DNA templates and primers.
