Two-Step RT-PCR Kit
User’s Instruction
Introduction:
This Two-Step RT-PCR Kit has all the reagents needed to synthesize the
first-strand cDNA followed by amplification of the cDNA product using PCR.
Oligo(dT)18 and random nonamer primers (dN)9 are provided according to
different experimental needs. The Kit uses M-MLV Reverse Transcriptase,
which is the protein product of mouse leukemia virus gene pol with a single 71
kDa peptide chain. The enzyme has a low RNase H activity. Using oligo(dT)18
or random nonamer as the primer, M-MLV RTase synthesizes the first-strand
cDNA. PCR reagents provided in this kit are then used to amplify the cDNA.
This kit provides a complete solution from RNA to PCR products, simple and
convenient for use.
Package and Components:
25 reactions
1. M-MLV Reverse Transcriptase (200U/µl)
2500U/12.5 µl
2. 5×M-MLV Buffer
100 µl
3. 100 mM DTT
50 µl
4. dNTPs(10 mM each)
37.5 µl
5. RNasin (40U/µl)
250U/6.25 µl
6. Oligo(dT)18 (20 pmol/µl)
0.5 OD/tube
7. (dN)9 (20 pmol/µl)
0.5 OD/tube
8. DEPC-treated water
1.5 ml
9. Taq DNA polymerase (5U/µl)
50U/10 µl
10. 10×PCR Buffer
50 µl
Procedures：
1. Reverse transcript reaction
(1) Mix the followings in a sterile thin-wall microtube:
Total RNA
0.5~1µg
(dN)9 or oligo(dT)18
100pmol
(2) Denature the mixture at 70℃ for 5min, and cool the tube on ice
rapidly, then add the followings sequentially:
5×M-MLV Buffer
dNTPs(10 mM each)
RNasin
100 mM DTT
M-MLV Reverse Transcriptase
DEPC-treated water
4 µl
1 µl
10 U
2 µl
100 U
up to 20 µl
(3) Mix the solution and centrifuge briefly, then incubate for 1 hr at the
appropriate temperature: 42℃ for oligo(dT)18 primers, and 37℃ for
(dN)9 primers.
(4) Stop the reaction by incubating at 94℃ for 5 min and cool the tube on
ice.
(5) The cDNA synthesized using this system can be used directly in PCR
amplification or other downstream applications.
2. PCR amplification
(1) Mix the followings in a sterile thin-wall microtube:
Synthesized cDNA
10×PCR Buffer
Forward primer
Reverse primer
dNTPs(10 mM each)
U-Taq DNA Polymerase
ddH2O
1-3 µl
2 µl
10 pmol
10 pmol
0.5 µl
1.5 U
up to 20 µl
(2) Mix the solution and centrifuge briefly, then begin the PCR
amplification:
Predenature the template at 94℃ for 2.5min. Then enter the following cycles
for 30~40 times (this amplification parameter is for reference only): 94℃ 30s,
60℃ 45s, 72℃ 1~3min. Extend at 72℃ for 7min lastly.
Remarks:

Ensure the integrity and purity of your RNA. The quality of RNA is the key
for first-strand cDNA synthesis. The integrity and purity of RNA can be
inspected by the ratio of OD260/OD280 and agarose gel analysis. The
common ratio of purified RNA is 1.8~2.0, if not, the RNA should be purified
further. The ratio of eukaryotic RNA 28S/18S is about 2:1, if not, the RNA
has been degraded.

Avoid RNase contamination. All vessels, reagents and solutions must be
sterile, and all procedures must be carried out with gloves.

Select the right primers for first-strand cDNA synthesis. Primer
oligo(dT)12-18 can ensure the synthesized cDNAs have intact 3’-end, and
get the first strand cDNA close to full length. Primer (dN)6 or (dN)9 can
anneal to many sites of the mRNA, and produce short length first strand
cDNA segments, which is often used to acquire 5’-end sequence and to
obtain cDNA from RNA with secondary structure or stop site that stops the
reverse transcription.
Troubleshooting Guide
1. Why the yield of cDNA is low?
Possible causes: (1) The quality of template RNA was too low. (2) The
concentration of RNA was estimated too high. (3) Reverse transcriptase
inhibitor existed or reverse transcriptase was insufficient. (4) Reaction volume
was too large. The common volume should not be more than 50µl.
2. Why the long cDNA can’t be synthesized?
(1)RNA has been degraded: all vessels and reagents should be sterile and
treated with DEPC to avoid RNase. At the same time, RNase inhibitor should
be added into the reverse transcription reaction. (2) Improper reaction
condition: condition should be optimized, including quantity of reverse
transcriptase, salt concentration, reaction temperature (37~56 ℃ ) and
concentration of DTT (0.5~10mM). (3) Secondary structure of RNA: increase
reaction temperature or use random primer.