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MBT076 Hi-cDNA Synthesis Kit

Product Name Product Code Kit Packing

MBT076-10R 10 reactions
Hi-cDNA Synthesis Kit MBT076-25R 25 reactions
MBT076-100R 100 reactions
Description:
Hi-cDNA Synthesis Kit is designed for reverse transcription where cDNA (complementary DNA) is synthesized
in-vitro from an mRNA template by an enzyme that has reverse transcriptase activity. Moloney Murine
Leukemia Virus Reverse Transcriptase (M-MuLV RT) is an RNA-dependent DNA polymerase that is used in
cDNA synthesis. This step is very important in order to perform PCR since DNA polymerase can act only on
DNA templates. The resulting cDNA is single-stranded and this process is called reverse transcription (RT) or
first strand cDNA synthesis. The kit contains Random Hexamer, Oligo (dT) and Random Hexamer : Oligo(dT)
Mix along with Ribonuclease Inhibitor.

Random Hexamers are commonly used for priming single-stranded DNA or RNA for extension by DNA
polymerases or reverse transcriptases.

Oligo(dT) is single-stranded sequence of deoxythymine (dT), used for priming reactions catalyzed by reverse
transcriptase. The transcript is primed in the poly(A) tail of mRNA molecules.

Ribonuclease Inhibitor are commonly used as a precautionary measure in cDNA synthesis to inhibit
ribonucleases (RNases) that can sometimes co-purify with isolated RNA and compromise downstream
applications.

Hi- cDNA Synthesis Kit is provided with:

Reagents provided for (reactions)
Components Product Code
10R 25R 100R
RT Buffer for M-MuLV DS0279 50 µL 125 µL 500 µL
10X Solution for M-MuLV DS0280 25 µL 62.5 µL 250 µL
M-MuLV Reverse Transcriptase (RNase H-) MBT072 12 µL 30 µL 120 µL
Ribonuclease Inhibitor DS0921 7 µL 17.5 µL 70 µL
Random Hexamer DS0146 15 µL 37.5 µL 150 µL
Oligo (dT) DS0145 15 µL 37.5 µL 150 µL
Random Hexamer : Oligo(dT) Mix DS0922 25 µL 62.5 µL 250 µL
10 mM dNTP Mix MBT078 25 µL 62.5 µL 250 µL
Molecular Biology Grade Water for PCR ML065 500 µL 1.25 mL 5 mL

Storage and Stability

Store the Hi-cDNA synthesis Kit at –20°C in a constant-temperature freezer. When stored under these
conditions, the kit components are stable for 1 year.

Registered Office
HiMedia Laboratories Pvt Ltd.
WHO Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Fax: 6147 1920
15
GMP Thane, (West) 400604, Maharashtra, INDIA. Web: www.himedialabs.com
CERTIFIED Customer Care No.: 00-91-22-6116 9797 Email : [email protected]
Tel: 00-91-22-6147 1919, 6903 4800 [email protected]

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made
or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein
Materials needed but not provided:
 Thermal cycler
 PCR tubes (Product code PW1255) or PCR Strips (Product code: PR17) or PCR Plates (Product code:
PR2 / PR3 / PR19)
 Barrier Micropipette Tips (Product Code: LA749 / LA749A / LA751 / LA751A / LA750 / LA750A / LA859
/ LA859A)
 Micropipettes

Procedure
1. Add the reagents as follows:
Volume per reaction
Ingredients Code Random Random
Oligo(dT)
Hexamer Hexamer:Oligo(dT) Mix
Random Hexamer* DS0146 1 µL - -
Oligo(dT)* DS0145 - 1 µL -
Random Hexamer : Oligo(dT) Mix* DS0921 - - 2 µL
RNA template - 5 ng to 5 µg
Molecular Biology Grade Water for PCR ML065 Up to 10 µL
*Alternatively, one can use a gene-specific reverse transcription primer.

2. Incubate for 5 min at 65°C, then cool immediately on Ice.

3. Prepare the reaction mixture in a total volume of 20 µL.

Ingredients Volume per reaction
Code
Template RNA Primer Mixture (from step 2) 10 µL
RT Buffer for M-MuLV DS0279 4 µL
10X Solution for M-MuLV DS0280 2 µL
M-MuLV Reverse Transcriptase (RNase H-) MBT072 1 µL
Ribonuclease Inhibitor DS0920 0.5 µL
10 mM dNTP mix MBT078 2 µL
Molecular Biology Grade Water for PCR ML065 Up to 20 µL

4. Gently mix and ensure that all the components are at the bottom of the amplification tube. Centrifuge
briefly if needed.

5. For preparation of cDNA using, incubate the complete reaction mix as follows:
a. For preparation of cDNA using, incubate the complete reaction mix using Random Hexamer
Random Hexamer No. of cycles
25°C for 5 minutes 1 cycle
42°C for 60 minutes 1 cycle
70°C for 5 minutes 1 cycle
Hold at 4°C optional

OR
b. For preparation of cDNA using, incubate the complete reaction mix using For Oligo(dT)
Oligo(dT) No. of cycles
42°C for 60 minutes 1 cycle
70°C for 5 minutes 1 cycle
Hold at 4°C optional

2
 OR
c. For preparation of cDNA using, incubate the complete reaction mix using Random Hexamer
: Oligo(dT) Mix
Random Hexamer:Oligo(dT) Mix No. of cycles
25°C for 5 minutes 1 cycle
42°C for 60 minutes 1 cycle
70°C for 5 minutes 1 cycle
Hold at 4°C optional

6. The cDNA can be further used to perform conventional or Real-Time PCR assay.

Amplification Data
M 1 2 3 4 5 6
Lane Sample
M 100bp DNA ladder
1,2 Amplicon obtained using
Random Hexamer
4,5 Amplicon obtained using
Oligo(dT)
3,6 Negative controls

Representative data of Semi-quantitative PCR of cDNA synthesized using Hi-cDNA Synthesis Kit (MBT076) after amplification

Ct value Tm

17.01 85.2

16.64 85

16.7 84.9

Representative data of Real-time SYBr based PCR of cDNA synthesized using Random hexamer : Oligo (dT) mix of Hi-cDNA
Synthesis Kit (MBT076) after amplification

Quality control
Detected free of RNases, endonuclease and exonuclease activities.

Warning
Not for Medicinal Use

Precautions
Read the procedure carefully before starting the experiment. Wear protective gloves/protective clothing/eye
protection/face protection. Follow good clinical laboratory practices while handling clinical samples. Standard
precautions should be followed as per established guidelines. Safety guidelines may be referred in safety data
sheets of the product.
3
 Troubleshooting Guide

Sr. No. Problem Possible cause Possible solution

No cDNA synthesis
For the cDNA synthesis step, incubate <50°C.
(temperature too high)
RNase contamination Maintain aseptic conditions
Not enough starting
Increase the concentration of template RNA
template RNA
RNA has been damaged or
Replace RNA if necessary
degraded
RT inhibitors are present inRemove inhibitors in the RNA preparation by an
No amplification
1 RNA additional 70% ethanol wash.
product
Note: Inhibitors of RT include SDS, EDTA,
guanidium salts, formamide, sodium phosphate
and spermidine
Annealing temperature is Decrease temperature as necessary
too high
Extension time is too short Set extension time for at least 60 seconds per kb
of target length
Cycle number is too low Increase cycle number
Reaction conditions not  Optimize magnesium concentration
optimal  Optimize the primer
 Optimize the annealing temperature and
2 Low specificity extension time
Oligo(dT) or Random Use only gene-specific primers
primers used for first-
strand synthesis
Contamination by genomic  Pretreat RNA with DNase I
DNA
Unexpected
Nonspecific annealing of  Vary the annealing temperature
bands after
3 primers  Optimize the magnesium concentration for
electrophoretic
each template
analysis
Primers formed dimers Design primers without complementary
sequences at the 3´ ends

Safety Information
The Hi-cDNA Synthesis Kit is for laboratory use only, not for drug, household or other uses. Take appropriate
laboratory safety measures and wear gloves when handling.

Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this
product. Follow established laboratory procedures in disposing of infectious materials and material that comes
into contact with clinical sample must be decontaminated and disposed off in accordance with current
laboratory techniques.

Please refer disclaimer Overleaf.

4
 Technical Assistance
At HiMedia, we pride ourselves on the quality and availability of our technical support. For any kind of technical
assistance, mail at [email protected].

-10°C Storage temperature

-20°C

Do not use if package is

damaged

HiMedia Laboratories Private Limited,

Reg. Off: Plot No. C-40, Road No. 21Y,
MIDC, Wagle Industrial Area, Thane,
(West) 400604, Maharashtra, INDIA.
Web: www.himedialabs.com

01/2025

PIMBT076_O/0122 MBT076-14

Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications. The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
for laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not
be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd. Reg.office : Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Thane, (West) 400604, Maharashtra, INDIA.
Customer Care No.: 00-91-22-6116 9797 Tel: 00-91-22-6147 1919, 6903 4800 Email: [email protected] Website: www.himedialabs.com