cDNA Labeling

Sigma offers many reagents for complementary DNA (cDNA) labeling. Gene expression analysis applications which might require cDNA labeling include microarrays, in situ hybridization, or Northern blotting. Probes may be radioactively or fluorescently labeled. For radioactive probes, often a 32P labeled nucleotide is incorporated during synthesis.

Fluorescently labeled cDNA may be directly incorporated during synthesis or post synthesis. Aminoallyl labeling is a post synthesis labeling method that avoids the enzymatic issues associated with the direct incorporation of nucleotides with large marker molecules. Aminoallyl labeling is accomplished by incorporating either aminoallyl-dUTP (A0410) or aminoallyl-UTP (A5660) at the time of cDNA synthesis. After synthesis and reaction clean-up (S5059), the small aminoallyl moiety is used to attach a molecular marker via a reaction with a NHS ester group on the marker.

cDNA Synthesis may be initiated by two types of available primers, hexanucleotide primers (H0268) for random priming or anchored Oligo(dT) (O4387) for priming off poly(A) tails. To prevent RNase activity from degrading RNA before and during cDNA synthesis reactions, Sigma offers diethyl pyrocarbonate (D5758), a recombinant ribonuclease inhibitor (R1158), and human ribonuclease inhibitor (R2520).

Other essentials offered for cDNA labeling include M-MLV Reverse Transcriptase (M1302), Deoxynucleotide (dNTP) sets 100 mM (DNTP100), and 10 mM (DNTP10).

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A0410 Aminoallyl-dUTP sodium salt ≥85%, lyophilized powder
A5660 Aminoallyl-UTP sodium salt ≥75%, lyophilized powder
DNTP100 Deoxynucleotide Set, 100 mM Individual dNTPs for routine PCR; 0.25 mL each
DNTP10 Deoxynucleotide Set, 10 mM Individual dNTPs for routine PCR; 0.5 mL each
D5758 Diethyl pyrocarbonate ≥97% (NMR)
H0268 Hexanucleotide Primers lyophilized powder
M1302 M-MLV Reverse Transcriptase Moloney Murine Leukemia Virus enzyme & buffer for cDNA synthesis
O4387 Oligo(dT)23, Anchored 70 μM in H2O
R1158 Ribonuclease Inhibitor recombinant, expressed in E. coli, 20-40 units/μL
R2520 Ribonuclease inhibitor human Inhibits ribonucleases during transcription and translation experiments
S5059 SigmaSpin Sequencing Reaction Clean-Up post-reaction clean-up columns
W4502 Water Nuclease-Free Water, for Molecular Biology