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ADVANCED™ 1 STEP RT-PCR KIT

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ADVANCED™ 1 Step RT-PCR KIT
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介绍详情

Ordering Information: 

CAT.NO.

Quantity

Specification

801-101-ZR

50 REACTIONS

RT PCR - 1 STEP

801-101-AR

100 REACTIONS

RT PCR - 1 STEP

801-101-ER

500 REACTIONS

RT PCR - 1 STEP

Product Description: 

WISENT ADVANCED 1 STEP RT PCR MIX uses the latest developments in reverse transcriptase technology and buffer chemistry for efficient cDNA synthesis and PCR in a single tube. Proprietary small-molecule hot-start technology offers improved specificity and sensitivity compared to other methods.Our Taq HS DNA Polymerase can perform consistently well on a broad range of templates including both GC and AT rich. We use Thermostable reverse transcriptase 45°C to 55°C and advanced RNAse inhibior.

Take advantage of our Proprietary hot-start technology for unrivalled detection of low copy number templates.

 

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Product Stability And Storage:

On arrival store at -25°C to -15℃.

Avoid prolonged exposure to light.

If stored correctly the mix will retain full activity for 12 months. It is stable at 2-8°C for 1 month and can go through 30 freeze/thaw cycles with no loss of activity.

Instructions For Use:

OUR KIT CONTAINS :

1.) 2 x mix containing HS Taq polymerase, MgCl2, dNTPs, enhancers and stabilisers. The buffer composition is optimal, do not add any other enhancers or MgCl2.

2.) 20 x RTase that also contains an RNAse inhibitor. IT IS IMPORTANT TO USE THE CORRECT VOLUME. THE VOLUME IS CRITICAL TO THE RESULT.

REMARKS:

1.) Primers should have a predicted melting temperature of around 60°C using default primer 3 settings.

2.) Reverse transscription: an incubating temperature of 45°C . For 10 minutes is recommended except where region of interest contain high secondary structure incubation in which case up to 55 °C might be used. For aplicons above 1 kb, the incubation time should be increased to 20 min.

3.) Determine the optimal annealing temperature experimentally by performing a temperature gradient.

4.) Optimal extension is achieved at 72 °C . Optimal extension time depends on amplicon length and template. Normally 15 seconds per kilobase is used for amplification for aplicons between 1 kb and 3 kb.

Reaction Setup:

1Before starting, briefly vortex 2x WISENT ADVANCED 1 STEP MIX.

2 Prepare a mastermix, as follows:

Add

Volume

Final concentration

ADVANCED 1 STEP MIX

25 ul

1X

Forward primer (10 uM)

2.0 ul

400 nM

Reverse primer (10 uM)

2.0 ul

400 nM

20 X RTase

2.5 ul

1 X

Template RNA

total RNA ：1 pg to 1ug
mRNA ：> 0.01 pg mRNA

variable

Add PCR grade water up to 50 ul final volume

3 Program your instrument using following conditions:

Cycles

Temperature

Time

Notes

1

45 °C - 55 °C

10 min.

45 °C is recomended for most applications, see above

1

95°C

2 minutes

Polymerase activation

40

95 °C

10 seconds

Denaturation

60-65 °C

10 seconds

Anneal

1

72°C

30-60 seconds

15 sec. Per kb

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