Thermo Scientific
RevertAid Reverse Transcriptase
EP0441
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For performing the microarray assay, we need to label the cDNA with a fluorescent nucleotide. For this, we need a robust reverse transcriptase. RevertAid transcriptase is a recombinant MMLV RT enzyme with RNAse H and DNA dependent polymerase activity as an extra feature with a wide range of optimum temperatures.
cDNA conversion/labelling the cDNA with fluorescent nucleotide
Total RNA
Around 5 micrograms of total RNA is used to prepare the cDNA. 0.5 microgram of the oligo dt primer is added and then made up to 12 microliters with NF water and incubated at 60 degrees. After the incubation step, 4 microliters of the 5X RT buffer is added along with dNTPs and finally, 1 microliter of the RT enzymes are added and allowed to incubate at 42 degrees. At the end, the sample is heated to 70 degrees for 5 minutes to inactivate the enzyme.
Always use good quality RNA as the starting material.
A very good quality of the cDNA is obtained with longer than 13kb in length.
N/A
Ease of optimization
Costly
If you are looking for an efficient cDNA labeling RT kit, I strongly recommend this kit.