Easy-Go RT Premix
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 tubes2. Oligo (dT)18 / 0.5 OD (16.5μg)
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 / 20 µl reactionTemplate
RNA / Total RNA / 0.5-1.0 µg
Poly(A)+ RNA / 0.05-0.1 µg
Primer / Sequence specific / 10-30 pmol
Oligo dT18 / 100 pmol
Random primer
/ 100 pmol2. 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.