Download R111-HiScript™ 1st strand cDNA Synthesis Kit重翻

HiScript® 1st strand cDNA Synthesis Kit
Catalog # R111
Introduction
HiScript® Reverse Transcriptase is a brand new reverse transcriptase based on mutagenesis of
M-MLV (RNase H-) Reverse Transcriptase. HiScript® Reverse Transcriptase is most active at
50°C. It can tolerate 55°C reaction temperature and is applicable to reverse transcription of
RNA templates with secondary structures. Elevated affinity of HiScript® Reverse
Transcriptase to the template makes reverse transcription reaction more efficient, with more
full-length cDNA can be obtained and as low as 1 pg total RNA can be detected, which is
especially applicable to a small amount of templates and the reverse transcription of low-copy
genes. In addition, the mismatch rate of HiScript™ Reverse Transcriptase is lower than that of
M-MLV (RNase H-) Reverse Transcriptase, thereby increasing the fidelity of cDNA cloning.
Based on HiScript® Reverse Transcriptase, HiScript® 1st strand cDNA Synthesis Kit contains
all components needed to synthesize high-quality 1st strand cDNA. Reaction products are
applicable to subsequent PCR, qPCR and PCR cloning.
2× RT Mix contains optimized buffer system and dNTP. This Mix contains HiScript® Reverse
Transcriptase and RNase inhibitor. Oligo (dT)18 and Random hexamers can be chosen as
primers for reverse transcription as needed.
Package Information
Components
R111-01 25 rxn (20μl /rxn)
R111-02 50 rxn (20μl /rxn)
RNase free ddH2O
1 ml
1 ml
2 × RT Mix a
HiScript™ Enzyme Mix b
Oligo (dT)18 (50μM)
Random hexamers (50 ng/μl)
a Contain 1 mM each dNTP.
b Contain RNase inhibitor.
250μl
50μl
25μl
25μl
500μl
100μl
50μl
50μl
Storage
Store at -20℃.
Quality Control
None of the components is contaminated by exonuclease, exonuclease or RNase after testing.
Functional test 1: Taking 500 ng Hela cell total RNA as template and Oligo dT18 as primer, react
for 30 min at 50℃. Take 1/10 of cDNA products to carry out PCR amplification of DNCH gene.
After agarose gel electrophoresis and EB staining, a clear 5.6 kb band can be detected..
Functional test 2: Taking 100 pg Hela cell total RNA as template and Oligo dT18 as primer, react
for 30 min at 50℃. Take 1/10 of cDNA products to carry out PCR amplification of β-actin gene.
After agarose gel electrophoresis and EB staining, a clear 550 bp band can be detected.
Protocol
Materials needed to be prepared by users
 RNase free microcentrifuge tube of 1.5 ml or 0.2 ml PCR tube
 Water bath or PCR instrument
 Ice
RNA
 High-quality and integrate RNA is of critical importance to obtaining high-quality cDNA.
HiScript™ 1st strand cDNA Synthesis Kit is applicable to reverse transcription of 1 pg to 2.5
μg total RNA.
 RNase inhibitor is already contained in HiScript™ Enzyme Mix, which is used to resist trace
RNase that might exist in the environment or system.
Synthetic condition of 1st strand cDNA
 All operations should be performed on ice.
 For GC-rich templates or those with complex secondary structures, the temperature for
synthesis of cDNA 1st strand can be increased to 55℃.
Choosing primers
Synthesis of 1st strand cDNA can be carried out by choosing Random hexamers, Oligo (dT) 18 or
gene specific primer (GSP) as primers.
Random hexamers is of lowest specificity. All RNA, including mRNA, rRNA and tRNA can
be templates of Random hexamers. When the target area of RNA has complex secondary
structure or is GC-rich, and Oligo (dT) 18 or gene specific primers (GSP) cannot
effectively synthesize cDNA, Random hexamers can be used.
 Oligo (dT) 18 hybridizes at high efficiency to the 3´ poly (A) region present in most mature
eukaryotic mRNA. It is the first choice for most cases and generally the highest
consistency can be obtained.
 Gene specific primer (GSP) has the highest specificity. But under some circumstances,
GSP used for PCR reaction cannot effectively synthesize cDNA. Then use Oligo (dT) 18
instead and try again.
Guidelines for PCR
Products of 1st strand cDNA can be directly used as templates for PCR reaction. It is suggested
that the volume of cDNA as templates should not exceed 1/10 of the volume of PCR reaction. In
accordance with different experimental objectives, the following DNA Polymerase is
recommended:
AceTaq™ DNA Polymerase. It is a chemically modified hot-start Taq enzyme with the highest
sensitivity, and is recommended to amplify low-copy gene from cDNA.
 Phanta™ Super Fidelity DNA Polymerase. It is a high-fidelity DNA polymerase with the
highest fidelity and extremely high amplification efficiency, and is recommended to amplify DNA
for cloning.
 LAmp™ DNA Polymerase. It is recommended to perform long fragment PCR, and the
amplicon length can reach up to 15kb.
Guidelines for reverse transcription reaction
Set up the following mixture in RNase free centrifuge tube
RNase free ddH2O
to 20 μl
2 × RT Mix
10 μl
Hi Script™ Enzyme Mix
2 μl
Oligo (dT) 18 (50 μM)
1μ
or Random hexamers (50 ng/μl)
or Gene Specific Primers (2 μM)
Total RNA: 100 pg-5 μg Poly (A)+
RNA: 10 pg-500 ng
Template RNA
Carry out the first strand cDNA synthesis according to the following conditions:
Use Oligo (dT)18
50℃
45 min*
85℃
5 min
Use Random hexamers
25℃
10 min
50℃
45min*
85℃
5min
Use Gene Specific Primers
50-55℃
45min*
85℃
5min
*May
be adjusted within 30-60 min. Prolonging reaction time may help to obtain longer cDNA
(>5 kb).
HiScript™ Reverse Transcriptase can be deactivated by incubation at 85℃ for 5 min. cDNA
can be stored at -20℃ or be immediately used in PCR reaction.
Troubleshooting
Problems and Cause
Possible solutions
No PCRproducts
RNase contamination
There are inhibitors of
transcription reaction in RNA
Besides using RNase free tip and centrifuge tube,
it is recommended that the reagents used are
exclusive for RNA experiments and the
operations should be performed at independent
and clean area. Make sure to wear gauze masks
and disposable gloves and avoid talking. After
total RNA is extracted, it is recommended to take
a small amount to carry out electrophoresis on
1% agarose gel to confirm the integrity of RNA.
Taking mammalian cell/tissue as example, if total
RNA is intact, three bands can be seen clearly on
gel, whose molecular weights are 28s, 18s and 5s
respectively. If three bands can be seen but their
shapes are blurred or diffuse, then part of RNA
degrades. In this case, please start reverse
transcription immediately and properly increase
the amount of templates; if only one band with
small molecular weight or no band can be seen,
RNA has been completely degraded. In this case,
prepare new RNA.
reverse
Inhibitors of reverse transcription reaction include
phenol, salt, SDS, EDTA, etc. It is suggested to
carefully wash off RNA precipitation (Flick the
bottom of the tube to suspend the precipitation, and
leave it still for several minutes) with 75% ethanol
(set up with DEPC water).
There are complex secondary structures or
high GC content in the target area of
RNA.
Use Random hexamers as primers for 1st strand
synthesis. Elevate the reverse transcription
temperature to 55℃.
Non-specific amplification
Contamination of genomic DNA
Use Trizol total RNA extraction reagent (such as
RNA isolater, cat No. R401-01), add chloroform and
separate it by centrifuging. Avoid pipe the middle
layer when transfer the supernatant. Use primers that
hybridize both contiguous exons.
PCR conditions are not optimized
Use hot-start Ace TaqTM DNA Polymerase
Adjust annealing temperature and concentration of
Mg2+