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Protocol for First-strand cDNA Synthesis
(PCR template generation with M-MuLV Reverse Transcriptase)

The following protocol is optimized to generate first-strand cDNA for use in two-step RT-PCR.
Mix and briefly centrifuge all components after thawing, keep on ice.

1. Add into sterile, nuclease-free tube on ice in the indicated order:
   

   Template RNA	total RNA or poly(A) RNA or specific RNA	100 ng - 5 µg 10 - 500 ng 0.01 pg - 0.5 µg
   Primer	oligo(dT)18 or random hexamer or gene-specific	0.5 µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
   	DEPC-treated Water	to 11.5 µl

2. Optional. If RNA template is GC rich or is known to contain secondary structures, mix briefly, centrifuge briefly and incubate at 65°C for 5 minutes, chill on ice, briefly centrifuge and place on ice.
3. Add the following components in the indicated order:
   

   5X reaction buffer for Reverse Transcriptase	4 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   dNTP Mix, 10 mM each	2 µl (1 mM final concentration)
   M-MuLV Reverse Transcriptase	2 µl (40 u)
   Total volume:	20 µl

   Mix gently and centrifuge briefly.

4. If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 minutes at 37°C.
   If  random hexamer primer is used, incubate 10 minutes at 25°C followed by 60 minutes at 37°C.
   For transcription of GC rich RNA reaction temperature can be increased to 45°C
5. Terminate the reaction by heating at 70°C for 10 minutes. Do not heat-inactivate enzyme prior to analysis of long cDNA to avoid cleavage.

Note

• The reverse transcription reaction product can be directly used in PCR or stored at -20°C.
• Use 2 µl of the reaction mix to perform PCR in 50 µl volume.

Reference

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

Ordering Information

M-MuLV Reverse Transcriptase dNTP Mix, 10 mM each RiboLock™ RNase Inhibitor First Strand cDNA Synthesis Kit Primers Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP Taq DNA Polymerase (recombinant) Taq DNA Polymerase (native): without BSA with BSA 2X PCR Master Mix High Fidelity PCR Enzyme Mix Long PCR Enzyme Mix DNase I, RNase-free DNA Polymerase I, E.coli RNase H, E.coli T4 DNA Ligase Pyrophosphatase, Inorganic (from yeast) DNA ladders RiboRuler™ RNA ladders DEPC-treated Water

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Updated gegužės 28, 2009 09:15