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Bionexus RT-PCR Premix is optimized ready-to-use mixture of M-MLV
Reverse Transcriptase RNase H Minus, ExActTM Taq, reaction
buffer, dNTP mixture, stabilizer and tracking dye as 2 fold concentration.
It can be provided as 5X mastermix pre-aliquoted into each PCR tube,
thus PCR reaction simply can be preformed by only adding primers
and template DNA reducing any mistake and contaminations, and making
a high reproducibility with a various and large number of samples.
|
Contents
|
| Reaction Vol. Final Conc. |
20 ul
|
50 ul
|
| M-MLV Reverse Transcriptase |
120 U
|
300 U
|
| ExActTM Taq |
1.5 U
|
3.75 U
|
| Each dNTP (dATP, dCTP, dGTP, dTTP) |
400 µM
|
400 µM
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| MgCl2 |
1.5 mM
|
1.5 mM
|
| Stabilizer & tracking dye |
-
|
-
|
1. Heat the template RNA and primer at 65Cº for 5 min and
quick chill on ice (or perform after step 3)
2. Add the heated template RNA and primer
3. Add DEPC-treated sterile water up to 20 ul or 50 ul
4. Incubate at 42Cº (up to 55Cº) for 1 hr
5. Perform PCR
5.1. Heat denaturation at 96Cº for 3 min
5.2. PCR cycle (25~35 cycles)
94Cº 1 min
55Cº1 min
72Cº 1 min
5.3. Last extension at 72Cº for 5 min
6. Load PCR reaction samples on agarose gel and perform electrophoresis
|
Concentration of template RNA and
primer
|
|
|
50 ul reaction
|
| Total RNA |
1 ng - 5 ug
|
1 ng - 5 ug
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| mRNA |
1 ng - 0.5 ug
|
1 ng - 0.5 ug
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| Specific primer |
0.5 -0.1 uM
|
0.5 - 1 uM
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|
|
|
-20 Cº
 |
Fig.1. Amplification
of various cDNAs using Bionexus's RT/PCR Premix with human
A49 cell line total RNA. M: 1 kb ladder, Lane 1 : TGF-beta
2 (1258bp), Lane 2 : TGF-beta 3 (1262bp), Lane 3 : Phospholipase
2A (449 bp). |
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Fig.2. cDNA (HDPE) Amplification
of HeLa cells using RT/PCR Premix at different cDNA synthesis
temperature. M: 1 kb ladder, Lane 1 : 45 Cº, Lane 2 :
50 Cº |
 |
Fig.3. cDNA (TNFR) Amplification
of HeLa cells using RT/PCR Premix with serially diluted total
RNA from 1ug to 0.1ug M: Lambda Hind III |
| Products |
Cat#
|
Size
|
| Nexus RT-PCR Premix, 2x mastermix |
BNRTP700
|
1ml
|
| Nexus RT-PCR Premix, 5x mastermix,
prealiquoted, 96 tubes |
BNRTP500
|
20ulx96
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| Nexus RT-PCR Premix, 5x mastermix, prealiquoted,
96 tubes |
BNRTP501
|
50ulx96
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