SCRIPT RT-PCR Two-Step Kit

Two-Step RT-PCR Kit for highest sensitivity and specificity

Product Cat. No. Amount Price (EUR) Buy / Note
S pack PCR-506S 25 reactions x 50 μl 118,00 Add to Basket/Quote Add to Notepad
L pack PCR-506L 200 reactions x 50 μl 670,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid

Availability Restriction: Exclusively distributed in Japan by Greiner BioOne

Description:
SCRIPT RT-PCR Two-Step Kit is designed for maximum sensitive and specific RT-PCR. The kit is based on a genetically engineered RT polymerase with enhanced thermal stability resulting in an increased specificity, higher cDNA yield and an improved efficiency for highly structured and long cDNA fragments.
It contains all reagents required for RT-PCR (except template and primer) in one box combining simple handling with high flexibility. The premium quality polymerases, ultrapure dNTPs and the optimized complete reaction buffers ensure superior amplification results.
RT-PCR is used to amplify double-stranded DNA from single-stranded RNA templates. In the RT step the reverse transcriptase synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template. In the first cycle of the PCR step synthesis, Taq DNA polymerase synthesizes DNA molecules complementary to the cDNA, thus generating a double-stranded DNA template. During subsequent rounds of cycling the DNA polymerase exponentially amplifies the double-stranded DNA template.
Kit contents:
SCRIPT Reverse Transcriptase (red cap)
200 units/μl SCRIPT Reverse Transcriptase in storage buffer with,
50 % glycerol (v/v)
Hot Start Pol (red cap)
5 units/μl Hot Start Taq in storage buffer with 50 % glycerol (v/v)
dNTP Mix (white cap)
10 mM each dNTP (dATP, dCTP, dGTP, dTTP)
5x SCRIPT RT Buffer complete (green cap)
250 mM Tris-HCl (pH 8.3), 500 mM KCl, 30 mM MgCl2, 25 mM DTT
10x Hot Start Buffer complete (green cap)
200 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, pH 8.5 (25 °C)
RNase Inhibitor (yellow cap)
40 units/μl RNase inhibitor in storage buffer with 50 % glycerol (v/v)
RNase-free Water (white cap)

RT primer selection:
The RT step may be carried out using

  • gene specific primers for specific transcripts
  • oligo-dT10-25 for all polyA RNA (mRNA)
  • random hexamers or octamers for amplifying total RNA


Sensitivity:
Targets can generally be detected from 10 pg to 500 ng mRNA or 10 pg to 1 μg total RNA. Even lower amounts of RNA may be successfully amplified by using gene specific primers or highly expressed transcripts.
Recommended protocols for RT-PCR:

First Step (RT- Reverse Transcription)

1a Assay set-up without sample denaturation
(standard RNA/primer combinations)

Assay preparation
Add the following components to a nuclease-free microtube. Pipett on ice and mix the components by pipetting gently up and down. In general, water, RNA and primers should be mixed together before the remaining components are added.

componentstock conc.final conc.20 μl
assay
RNase-free water--fill up to
20 μl
RNA template-total RNA:
10 pg - 5 μg
or mRNA:
10pg-500ng
x μl
Primer10 μM-gene-specific primer:
10-20 pmol
(50-100 ng)
-oligo-dT15-25 primer:
50 pmol
(300 ng)
-random primer:
50 pmol
(100 ng)
- 1-2 μl
- 5 μl
- 5 μl
SCRIPT RT
Buffer
complete
5x1x4 μl
dNTP Mix10 mM each500 μM
each
1 μl
DTT stock
solution1)
100 mM5 mM1 μl
RNase Inhibitor2)40 units/μl40 units1 μl
SCRIPT Reverse Transcriptase3)200 units/μl100-200 units0.5-1 μl
1)Adding of up to 5 mM DTT may increase the yield and is recommended for individual optimization.
2)Addition of 20-40 units RNase inhibitor per assay is recommended and may be essential when working with low amounts of starting RNA.
3)100 units (0.5 μl) of enzyme is recommended for standard assays but increasing the amount of enzyme to 200 units (1 μl) per assay may show even higher transcription yields under selected assay conditions.

1b Assay set-up with sample denaturation
(RNA/primer with a high degree of secondary structure)

Preparation of the RNA Template / Primer Mix
Add the following components to a nuclease-free microtube and mix by pipetting gently up and down:

componentstock conc. final conc.20 μl
assay
RNase-free water--fill up to
10 μl
RNA template-total RNA:
10 pg - 5 μg
or mRNA:
10pg-500ng
x μl
Primer10 μM-gene-specific primer:
10-20 pmol
(50-100 ng)
-oligo-dT15-25 primer:
50 pmol
(300 ng)
-random primer:
50 pmol
(100 ng)
- 1-2 μl
- 5 μl
- 5 μl

Denaturation and primer annealing
Incubate the mixture at 65-70 °C for 5 min and place it at room temperature (if using specific primer) or on ice (if using oligo-dT or random primer).

Preparation of the Reaction Mix
Add the following components to a further nuclease-free microtube and mix by pipetting gently up and down:

componentstock conc.final conc.20 μl
assay
RNase-free water--fill up to
10 μl
SCRIPT RT
Buffer
complete
5x1x4 μl
dNTP Mix10 mM each500 μM
each
1 μl
DTT stock
solution1)
100 mM5 mM1 μl
RNase Inhibitor2)40 units/μl40 units1 μl
SCRIPT Reverse Transcriptase3)200 units/μl100-200 units0.5-1 μl
1)Adding of up to 5 mM DTT may increase the yield and is recommended for individual optimization.
2)Addition of 20-40 units RNase inhibitor per assay is recommended and may be essential when working with low amounts of starting RNA.
3)100 units (0.5 μl) of enzyme is recommended for standard assays but increasing the amount of enzyme to 200 units (1 μl) per assay may show even higher transcription yields under selected assay conditions.

Complete Reaction Mix
Add 10 μl Reaction Mix to 10 μl RNA Template / Primer Mix to complete the 20 μl Reaction Mix. Pipett on ice and mix by pipetting gently up and down.

2 First-strand cDNA synthesis
Incubation
Incubate the reaction mix at 50 °C for 30-60 min if using gene-specific primers. If using oligo-dT or random primers incubate at 42 °C for
10 min followed by incubation at 50 °C for 30-60 min.
[Please note: The optimal time depends on the length of cDNA. Incubation of 60 min is recommended for cDNA fragments of more than 2,000 bp length. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 55 °C for difficult templates with high secondary structure. Note that optimal reaction time and temperature should be adjusted for each particular RNA.]


Second Step (PCR)
Preparation of the PCR mix
Add the following components to a nuclease-free microtube and vortex gently:

componentstock conc.final conc.1 assay
RNase-free water4)--fill up to
50 μl
Hot Start Buffer complete10x1x5 μl
dNTP Mix10 mM each200 μM
each
1 μl
forward PCR Primer10 μM300-400 nM1.5-2 μl
reverse PCR Primer10 μM300-400 nM1.5-2 μl
Hot Start Pol5 units/μl1.25 units0.25 μl
RT assays from First Step--2-5 μl
4) The volume of RNase-free water depends on the amount of RT reaction mix added to the assay in step 7. The total recommended volume of the PCR assay is 50 μl.

Thermal cycling
Place the vials in a PCR cycler and start the following program.

initial
denaturation
95 °C5 min1x
denaturation95 °C10 sec30-40x
annealing5)55-65 °C20 sec30-40x
elongation6)72 °C1 min/kb30-40x
final
elongation
72 °C1 min/kb1x
5) The annealing temperature depends on the melting temperature of the primers.
6) The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of 1,000 bp is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the annealing temperature may be necessary for each new template / primer combination.