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EnergicScript®
First Strand cDNA Synthesis Kits
Description
The
EnergicScript® cDNA Synthesis Kit includes all the necessary reagents
for cDNA synthesis to be used in qPCR. Either total RNA,
mRNA, viral RNA or in
vitro transcribed
RNA can be used as a template for reverse transcription.
The kit includes both random primers and oligo(dT)
primers. The user can choose either of these or
lternatively use gene specific primers. Quantitative PCR
(qPCR) is a useful technique for the investigation of
gene expression,viral load, pathogen detection, and
numerous other applications. When analyzing gene
expression or viral load, the RNA of interest first
needs to be reverse transcribed into cDNA. The EnergicScript® cDNA Synthesis Kit is intended for cDNA synthesis for
two-step quantitative reverse transcription-PCR (qRT-PCR)
applications, where amplicons are usually around 200 bp
in length. This kit is can be used in conjunction with
ShineProbe qPCR Kits or with any other qPCR kit suitable
for the application. Gene
pecific primers, random primer or oligo(dT) primers can
be used for the RT step.Using specific primers can
decrease background, whereas random primer and oligo(dT)
primers are useful if several different amplicons need
to be analyzed from a small amount of starting material.
1.
random primer: at non-specific points along an RNA
template. In this case, all RNA in a population are
templates for cDNA synthesis.
2.
oligo(dT)18:
at the 3'-end of poly(A)+ mRNA. In this case, only
mRNA with 3'-end poly(A) tails are templates for cDNA
synthesis.
3.
gene specific primer: at a primer-binding site.
Importants
Notes
1.
Multiple freeze/thaw of RNA should be avoided. Thaw and
keep control RNA on ice.
2.
It is recommended that the first strand cDNA synthesis
is carried out under conditions where Rnase
contamination has been eliminated. Pipette tips and
tubes should be treated with 0.1% diethylpyrocarbonate (DEPC)
(soak overnight in 0.1% aqueous solution of DEPC at 37°C, then
heat at 100°C
for
30 min and autoclave).
3.
Wearing gloves is highly recommended.
4.As
most users do not need to do control First Strand cDNA
synthesis, this kit does not include control RNA.
5.
Incubation at 40°C
will work for most templates,
but it can be optimized between 40°C
and 48°C
if necessary. Raising the temperature can be helpful if the
template has strong secondary structures. Higher temperature
can also improve specificity if gene-specific primers are
used. Incubation time of 30 min is sufficient in most cases.
If the target is located near the 5'end
of a long transcript and oligo(dT) priming is used, or the
target is rare, cDNA synthesis time can be extended up to 60
min.
6. A
separate RNA denaturation step is generally not
required, but it can be performed before cDNA synthesis
if the template RNA has a high degree of secondary
structure. The denaturation step, 5 min at 65°C,
should be performed before adding 2x RT buffer and RTase
to the reaction mix.
Component
Components
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ZK00804
(50 rnsX
20ul each)
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ZK00805
(100 rnx X
20ul each)
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2XRT
Buffer
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500ul
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1000ul
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random
primer
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50ul
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100ul
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oligo(dT)18
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50ul
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100ul
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RTase
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50ul
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100ul
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DEPC-ddH2O
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1ml
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1ml
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Shipping
and Storage
The EnergicScript Kit is shipped in gel ice or ice bag. Upon arrival,
store all kit components
at -20°C. When using the kit, the leftover thawed mix
can be refrozen
and stored at -20°C without affecting the performance
of the kit. The kit is stable for six months from the
date of packaging when stored and handled properly.
Reaction
Setup
Components
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Volume(ul)
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Comments
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2XRT
Buffer
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10
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Mix
thoroughly
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random
primer
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1
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Alternatively
oligo(dT) or a specific primer can be used.
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RTase
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1
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Includes
RNase inhibitor
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RNA(ul)
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X
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Max
1ug,usually
2-6ul
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DEPC -ddH2O (ul)
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Y
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Add
water to fill up to the final
reaction volume
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Total
Volume(ul)
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20
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Cycling
Protocol
1.Protocol
using
random
primer
25°C10min→40°C30
min→80°C2
min→4°C hold
2.Protocol
using oligo(dT)18
25°C10min→42°C30
min→80°C2
min→4°C hold
3.Protocol
using specific
gene primer
48°30
min→80°C2
min→4°C hold
Reference
1.
Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular
Cloning: A Laboratory Manual (2nd Ed.) Cold Spring
Harbor University Press, Cold spring Harbor, NY, 1989.
2.
Ausubel, F.M. et al. eds., Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., NY,
1997.
 First Strand cDNA Synthesis Kits manual
Related Link
Peptide
Synthesis
Chromas
Make
Antibody
Lab
Consumables
Gene
Synthesis
dNTP
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