タイトルまたは説明
Product:
Reverse transcription kit independent primer (third generation
enzyme, independent primer, genome removed) Cat. No.: F***2A
Price: $**0 for
*0ul***0T Storage conditions:
**0°C Product
components:
|
Components
|
Specification
|
|
RTase III Primer Flexible
All-in-One Mix
|
**0 μl
|
|
Oligo(dT)*0VN (*0
μM)
|
**0 μl
|
|
Random hexamers (*0
μM)
|
**0 μl
|
|
dsDNase
|
2x *0ul
|
|
*0× dsDNase
Buffer
|
**0ul
|
|
Nuclease-Free
Water
|
2×1 ml
|
Product
Introduction The Primer Flexible is a high-efficiency,
low-pollution, high-quality, single-strand cDNA synthesis premix.
It contains multiple components required for single-strand cDNA
synthesis, such as M-MLV GIII Reverse Transcriptase and its
reaction buffer, RNase inhibitors, dNTPs, etc., and the reaction
can be started by adding RNA template, primers and water. It is
very convenient to operate. Different types of reverse
transcription primers can be used flexibly for different
experimental designs to meet diverse experimental needs.
Oligo(dT)*0VN or Random hexamers or Gene Specific Primers can be
selected according to different experimental scenarios. Using this
reverse transcription premix, a maximum of *2 kb cDNA can be
obtained within *5 minutes.
RNA extracted from cells often contains
genomic DNA contamination. If it is not removed before reverse
transcription, genomic DNA and cDNA will be amplified
simultaneously during downstream qPCR reactions (especially when
the primers are designed on the same exon), thereby affecting the
accuracy of gene expression quantification. This kit uses dsDNase
to efficiently remove genomic DNA contamination. dsDNase can
specifically digest double-stranded DNA (dsDNA or DNA strands in
DNA-RNA hybrid strands) and is heat-sensitive, which can be
rapidly and irreversibly inactivated at the reverse transcription
temperature. Compared with the traditional method of using DNase
I to remove genomic DNA contamination, dsDNase does not require
the addition of EDTA for inactivation, which not only saves
experimental time, but also reduces the inhibition of reverse
transcription reaction.
Instructions
I. Total RNA
1. Prepare the following reaction system on
ice:
|
Reagents
|
Amount of Usage
|
|
Total RNAa
|
*0ng*1ug
|
|
dsDNase
|
1ul
|
|
*0× dsDNase
Buffer
|
1ul
|
|
Nuclease-Free
Water
|
To *0 ul
|
a. It is recommended to use RNA extracted by
the kit as a template.
2. Gently pipette to mix and
spin; 3. Incubate at *7℃ for 2 min to remove
genomic DNA contamination;
Note: If the genomic DNA contamination in the
RNA is serious, the *7℃ incubation time can be appropriately
extended to 5 min.
4. Incubate at *5℃ for 2 min to inactivate
dsDNase and place on ice.
First-strand cDNA
synthesis 1.Prepare the following reaction system on
ice:
|
reagents
|
Usage
(experimental group)
|
|
Experiment
(1) "reaction product
|
*0ul
|
|
RTase III Primer Flexible
All-in-One Mix
|
4 μl
|
|
Oligo(dT)*0VN (*0
μM)
|
1 μl
|
|
Or Random hexamers (*0
μM)
|
1 μl
|
|
Or Gene Speciï¬c Primers (*0
μM)
|
0.1 μl
|
|
Nuclease-Free
Water
|
To *0 μl
|
2. Mix by gentle pipetting and
centrifuge; 3. Incubate at *5℃ for *5
min;
Note: If the target RNA does not contain a
poly(A) structure, it can be pre-incubated at *5℃ for *0
min. 4. After the reaction, incubate at *5℃ for
5 min to terminate the reaction;
5. Place the obtained cDNA solution on ice
for subsequent experiments; or store it immediately at
**0℃. Note: 1. The subsequent experiment is a
cloning experiment. If the RNA comes from eukaryotes, only Oligo
(dT) VN is needed. Adding Random hexamers will reduce the yield
of full-length cDNA; if the RNA comes from prokaryotes, only
Random hexamers or Gene Speciï¬c Primers are
needed. 2. If the subsequent experiment is qPCR,
please add Oligo(dT) VN and Random hexamers at the same time to
obtain cDNA with uniform reverse transcription efficiency at
different positions of mRNA.
II. miRNA
Genome removal
1. Prepare the following reaction system on
ice:
|
reagents
|
Amount of usage
|
|
miRNA
|
*0 pg~**0 ng
|
|
dsDNase
|
1ul
|
|
*0× dsDNase
Buffer
|
1ul
|
|
Nuclease-Free
Water
|
To *0 ul
|
2. Mix by gentle pipetting and
centrifuge; 3. Incubate at *7℃ for 2 min to remove
genomic DNA contamination;
Note: If the genomic DNA contamination in RNA
is serious, the *7℃ incubation time can be appropriately
extended to 5 min. 4. Incubate at *5℃ for 2 min to inactivate
dsDNase and place on ice.
First-strand cDNA
synthesis 1. Prepare the following reaction system on
ice:
|
reagents
|
Usage
(experimental group)
|
|
Experiment
(1)" reaction product
|
*0ul
|
|
RTase III Primer Flexible
All-in-One Mix
|
4 μl
|
|
Stem-loop primer(5
μM)b
|
1 μl
|
|
Nuclease-Free
Water
|
To *0 μl
|
b. The recommended final concentration of
stem-loop primers is 0.*5 μM, which can be adjusted within the
range of 0.1~0.5 μM. 2. Mix by gentle pipetting and
centrifuge; 3. Incubate at *5℃ for *5
min;
4. After the reaction, incubate at *5℃ for
5 min to terminate the reaction;
5. Place the obtained cDNA solution on ice
for subsequent experiments; or store immediately at
**0℃. Design of miRNA stem-loop primers and qPCR
primers 1. Stem-loop RT primer design: Based on the
universal stem-loop structure, only the last 6 bases need to be
modified according to different miRNA sequences. The universal
stem-loop sequence is: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC;
2. Taking miR***5p as an example, its
sequence is CAUACUUCCUUACAUGCCCAUA. Just add the reverse
complementary sequence of the 6 bases at the 3' end of miRNA
after the universal stem-loop
sequence, i.e.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC
"TATGGG"; 3. qPCR upstream primer design: The remaining
part of the miRNA sequence after removing the 6 bases at the 3'
end is used as the upstream primer. For example, the upstream
primer of miR***5p is (note that U is changed to
T): CATACTTCCTTACATG. Check the Tm value of the
primer. If the Tm value is low, add GC at the 5' end to make the
Tm value close to *0℃. Therefore, the upstream primer of
miR***5p can be designed as:
GCCGCCATACTTCCTTACATG,
*0.3℃; 4. The downstream primer is universal, and
the sequence is GTGCAGGGTCCGAGGT;
5. After the primer is designed, the
specificity of the primer needs to be tested through a
preliminary test. Generally, a melting curve is required to
detect the specificity of the primer; at the same time, it is
best to perform electrophoresis on the PCR product to detect
whether the product is single (the product length is very short,
and more than 3% agarose gel is
required). Precautions
1. Before using all reagents, please gently
turn them upside down to mix them, try to avoid bubbling, and use
them after a short centrifugation;
2. To prevent RNase contamination, please
keep the experimental area clean;
3. Wear clean gloves and masks during
operation; 4. The consumables such as centrifuge tubes
and gun tips used in the experiment must be guaranteed to be
RNase-free.
