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  7. Invitrogen™ Second Strand cDNA Synthesis Kit

Invitrogen™ Second Strand cDNA Synthesis Kit

Catalog No. A48571
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A48571 200 Reactions
A48570 50 Reactions
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Catalog No. A48571 Supplier Invitrogen™ Supplier No. A48571
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Description

Invitrogen™ Second Strand cDNA Synthesis Kit is design to produce double-stranded cDNA using first strand reaction without the need for intermediate organic extraction or ethanol precipitation steps. This convenient single-tube format speeds up the synthesis procedure and maximizes cDNA recovery. The kit contains premixed components to reduce the number of pipetting steps necessary to complete the procedure.

InvitrogenTM Second Strand cDNA Synthesis Kit is design to produce double-stranded cDNA using first strand reaction without the need for intermediate organic extraction or ethanol precipitation steps. This convenient single-tube format speeds up the synthesis procedure and maximizes cDNA recovery. The kit contains premixed components to reduce the number of pipetting steps necessary to complete the procedure.

Features of the Second Strand cDNA Synthesis Kit include:

  • Efficient synthesis of full-length double-stranded cDNA
  • Fast—procedure completed in 1 hour
  • Convenient—pre-mixed components
  • Complete—includes residual RNA removal reagents

Applications

  • Full-length double-stranded blunt-end cDNA synthesis for cloning
  • Double-stranded cDNA library construction

Specifications

Content And Storage Contents:
• Second Strand Enzyme Mix, 4 x 1 mL
• 5X Second Strand Reaction Mix, 4 x 250 μL
• 0.5 M EDTA, pH 8.0, 4 x 1 mL
• RNase I, 10 U/μL, 4 x 500 μL
• Water, nuclease-free, 12 x 1.25 mL

Storage: -15 to -25°C
Format 200 reactions
Product Type cDNA Synthesis Kit
Quantity 200 reactions
For Use With (Application) Cloning
Final Product Type Double Stranded cDNA
No. of Reactions 200 Reactions
Reaction Format Separate Components

Frequently Asked Questions (FAQs)

What is the correct reaction temperature that I should use for second strand cDNA synthesis using the Second Strand cDNA Synthesis Kit?

The second strand reaction must be incubated at 16 degrees C to prevent spurious synthesis by DNA polymerase I. At higher temperatures, DNA polymerase I tends to strand-displace rather than nick translate.
When using the Second Strand cDNA Synthesis Kit, what steps do I need to take to properly prepare the second strand reaction mixture?

Dilute the first-strand reaction as outlined in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019413_SecondStrandcDNASynthesisKit_UG.pdf) on page 2 under Second Strand cDNA Synthesis step. Failure to properly dilute the first-strand reaction changes the pH of the second strand reaction and influences the activity of the 3'-5' and 5'-3' exonuclease activities of DNA polymerase I.
When using the Second Strand cDNA Synthesis Kit, how can I avoid incomplete RT inactivation?

Always perform the RT inactivation step after first-strand cDNA synthesis.
I am getting low yield of second strand cDNA product using the Second Strand cDNA Synthesis Kit. What is the reason?

The main reasons are incomplete RT inactivation, improper preparation of the second strand reaction mixture, and incorrect reaction temperature.