Easy-Go™ RT PreMix
User’s Instruction
Description
The Easy-Go™ RT-Premix is a new, powerful, ready-to-use RT preparation
supplied in one tube and lyophilized format. The kit contains M-MLV Reverse
Transcriptase for preparation of full-length first-strand cDNA from RNA
templates. Both oligo(dT) and random primers are included in this kit
depending on your preferences or applications. For reaction set-up, all you
have to do is to add RNA templates, primers and water. Resulting populations
of high quality cDNA can be used in downstream applications such as PCR
amplification.
Kit Contents
1. Easy-Go™ RT PreMix
25 tubes
2. Oligo (dT)18
0.5 OD (16.5
3. Random primers
0.5 OD (16.5μg)
)
Notes Before Use
1. RNA Integrity, Storage and Stability.
The integrity and quality of template RNA is critical for successful cDNA
synthesis. RNA may be degraded by ribonucleases. RNA undergoes a slow
hydrolysis in aqueous solutions. Frequent freeze-thaw cycles and neutral to
basic pH accelerate chemical cleavage of RNA. All these factors can affect the
integrity of RNA. To synthesize full-length first-strand cDNA from RNA
templates successfully, refer to the recommendations as follows:

Check the integrity of RNA on a denaturing agarose gel. The 28S rRNA
band of intact RNA should be twice as intense as the 18S rRNA band.

Store aqueous RNA working solutions in small aliquots at -80℃. For long
term stability, store RNA as a precipitate in 96% ethanol at -80℃.

Adjust the pH of aqueous RNA working solutions to 4~5 with a 5~10 mM
sodium citrate buffer.
2. Avoiding RNase Contamination
RNases are ubiquitous in the laboratory and in cells. To eliminate the risk of
RNase contamination, follow these precautions: RNA-related reagents must
be treated critically to avoid RNase contamination. Glassware should be
heated to 180℃ for 12 hours, or placed in 0.1% DEPC solution at 37℃ for 12
hours with vigorously mixing followed by autoclaving at 120℃ for 30 minutes to
remove the DEPC. Plasticware should be placed in 0.1% DEPC solution to
inactivate RNase. Wear gloves when handling reagents, equipment and
samples. Pipettors should be wiped with 80% ethanol or isopropanol before
RNA work.
3. Avoiding Genomic DNA Contamination
Even trace amounts of genomic DNA can be amplified in the PCR reaction
along with the target cDNA, which affects the specificity of cDNA amplification.
If genomic DNA contamination is detected in the RNA sample, it should be
removed by treating the RNA sample with RNase-free DNase I before
first-strand cDNA synthesis.
4. Primers
Gene specific primer, oligo(dT) primer or random primer can be used for the
cDNA synthesis. Gene specific primer is most specific but may not efficiently
prime the first-strand cDNA synthesis. Oligo(dT) can only be used to prime
mRNAs with 3’-poly(A)+ tails. Random primer can anneal to non-specific sites
along RNA templates and can prime all RNAs in the population.
Protocol
1. Mix the template RNA and primer in a sterile tube as indicated below:
Reaction volume
Template
Total RNA
RNA
Poly(A)+ RNA
Sequence specific
Primer
Oligo dT18
Random primer
20 µl reaction
0.5-1.0 µg
0.05-0.1 µg
10-30 pmol
100 pmol
100 pmol
2. Incubate the mixture at 70℃ for 5 min and place it on ice.
3. Transfer the incubated mixture to an Easy-Go™ RT PreMix tube, then
make up the reaction volume with DEPC-Water to 20 µl.
4. Dissolve the lyophilized blue pellet by vortexing, and briefly spin down.
5. Add mineral oil to each tube (this step is unnecessary when using a
thermal cycler with top heating).
6. Perform cDNA synthesis reaction as follows:
42℃, 60 min (cDNA synthesis)
94℃, 5 min (RTase inactivation)
If PCR is needed following RT reaction, perform the PCR reaction as follows:
1. Transfer 1~3 µl of the RT product (synthesized cDNA) to Easy-Do™ PCR
PreMix tube (20 μl reaction volume).
2. Perform PCR cycles according to the PCR condition.