avans Biotechnology Corp.
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Instruments
BenchMateTM Thermomixer
Cell Disrupter : AgileGrinder
ELISA-Microplate : AgileReader
FlexHandTM Automated Pipetting System
Pipettor
Power Li-Dry Bath
Spectrophotometer
SpotLiter
Thermal Cycling
Tissue Microarrayer
Tissue Mixer Mill
Vortex Mixer : Agile Vortex Mixer
Chemical
3:1 Agarose
G-418 (Aminoglycosidic Antibiotic)
High Resolution Agarose
LE Multi-purpose Agarose
Low Melting Point(LMP) Agarose
Consumable
Tips & Tubes
Reagent
DNA Extraction & Amplification Kit
Pre-stained Protein Marker
SYBRGreen Real Time PCR Premix 2X
Taq
SYBRGreen Real Time PCR Premix 2X (No ROX)
Product Description：
SYBRGreen Real Time PCR Premix 2X (No ROX) is an optimized ready-to-use mastermix containing unique reaction buffer, dNTP, Taq DNA polymerase and SYBR Green I. The user can easily use it to detect and quantify the expression of target genes via SYBR Green I Real Time PCR.

This product is for real-time PCR instruments from Roche, Bio-Rad, Ceheid, Corbett Research, Eppendorf. If you are using real-time PCR instruments form ABI, please purchase ROX reference dye separately.

Specification：100 x 50ul reactions (2 x 1.25ml)
Storage Conditions： -20°C .
Procedure：
1. The concentration of input DNA sample could be 0.05fg/ul - 500pg/ul depending on the real-time PCR instrument. No need to purify samples if they are RT-PCR products.
2. Take out SYBRGreen Real Time PCR Premix 2X (No ROX) from -20oC freezer and thaw it. Keep the tube on ice.
3. Reaction setup: (Add each component in order)

Real Time Premix 2x

12.5ul

PCR Forward Primer (10uM)

0.5ul

PCR Reverse Primer (10uM)

0.5ul

DNA Template

 2.0ul

H2O

9.5ul

Total

25.0ul

* Use 2ul H2O to replace DNA template for no template control
4. Real-time PCR program:

Temp.( oC)

Time (sec.)

Cycle

95

30

1

95
60

10
30

35- 45

5. Analyze amplification curve according to the manufacturers’ instruction manual of Real Time PCR instruments.
Guidelines for Real-time PCR primer design：
(1) The PCR product size is ideally to be 60- 200bp
(2) Length of primer: 18-30bp; GC%: 40-60%; Tm: 60-68oC
(3) At the 3’ end, it should be avoided to be T, or more than 3 sequential G or C. As for primer dimers, the ?G should be > -5.0 kcal/mol or less than 3 sequential complementary bases.
(4) Check PCR efficiency of each primer pair by Agarose gel.