How to Make cDNA for Molecular Cloning

 How to Make Cloning-Ready cDNA Using the Maxima H Minus Double-Stranded cDNA Synthesis Kit

If you're planning to clone a gene or study transcript expression in detail, you need a reliable method for converting RNA into stable, cloning-ready cDNA. The Maxima H Minus Double-Stranded cDNA Synthesis Kit from Thermo Scientific is a powerful system that allows you to go from total RNA to double-stranded cDNA efficiently—all in a single tube.

In this post, we’ll walk through the key steps of using the kit, share critical tips for RNA handling, and discuss how to analyze and purify your cDNA for downstream applications like plasmid cloning.

What’s Special About the Maxima H Minus Kit?

• Single-tube reaction: Both first and second strand synthesis happen in the same tube, no need for organic extraction or precipitation steps.

• Thermostable RT enzyme: The Maxima H Minus reverse transcriptase has high thermal stability (up to 60°C) and lacks RNase H activity.

• Versatile primer support: Works with oligo(dT)18, random hexamer, or gene-specific primers.

• High cDNA yield: Synthesizes cDNA up to 11 kb long from 0.5–20 µg total RNA or 50 ng–5 µg mRNA.

Step-by-Step Protocol Overview

Prepare Your First Strand Synthesis Reaction

You’ll need:

• Total RNA (0.5 µg to 20 µg) or mRNA (50 ng to 5 µg)

• Oligo(dT)18, random hexamers, or gene-specific primers

• Nuclease-free water

• Maxima H Minus First Strand Enzyme Mix

• 4X First Strand Reaction Mix

Steps:

1. Mix RNA (1ug - 20ug for total RNA; 50ng - 5ug for mRNA) + primer (1ul for oligo dT, 100 pmol; 1ul or variable for random hexamer, can use 0.5ug - 5ug total RNA when using random hexamer ) + water to 14 µL.

2. Heat at 65°C for 5 min, then chill on ice.

3. Add:

• 5 µL 4X First Strand Reaction Mix

• 1 µL First Strand Enzyme Mix

4. For oligo(dT) or gene-specific primers: Incubate at 50°C for 30 min.
   For random hexamers: Incubate 10 min at 25°C, then 30 min at 50°C.

5. Inactivate enzyme: 85°C for 5 min, then chill on ice.

Proceed to Second Strand Synthesis

You’ll need:

• 5X Second Strand Reaction Mix

• Second Strand Enzyme Mix

• Nuclease-free water

• 0.5 M EDTA (pH 8)

Steps:

1. Into the same tube, add:

• 20 µL First strand cDNA

• 55 µL Water

• 20 µL 5X Second Strand Mix

• 5 µL Second Strand Enzyme Mix

2. Incubate at 16°C for 60 min.

3. Stop reaction: Add 6 µL of 0.5 M EDTA (pH 8).

Residual RNA Removal & Purification

If total RNA was your input, residual RNA remains post-synthesis.

1. Add 10 µL RNase I (10 U/µL)

2. Incubate 5 min at room temp

3. Purify your double-stranded cDNA using:

• GeneJET PCR Purification Kit, or

• Phenol:chloroform extraction

Tip: Elute in ≤ 20 µL buffer for higher cDNA concentration.

RNA Quality: The Make-or-Break Factor

High-quality RNA is essential. Degraded RNA reduces yield and size of cDNA.

• Check integrity using agarose gel or a Bioanalyzer.

• Look for sharp 28S (~5 kb) and 18S (~2 kb) rRNA bands.

• Smears or missing bands indicate degradation—repeat extraction.

Primer Selection & Their Impacts

Primer Type	Targets	Use Case
Oligo(dT)18	Poly-A tail (mRNA)	Messenger RNA studies
Random Hexamers	All RNA species	Total RNA, non-coding RNA, degraded samples
Gene-Specific	Target sequence	Focused studies on one gene

Note: Higher random primer concentrations may reduce cDNA length.

Optional Control Reaction

The kit provides GAPDH poly-A mRNA and oligo(dT) primers for control testing. Follow the same synthesis protocol and run digested vs undigested cDNA on an agarose gel. Expect restriction fragment sizes of 615, 363, 227, and 143 bp if using FastDigest™ HinP1(Hin6I) restriction enzyme (#FD0484).

Downstream Applications

Purified blunt-end double-stranded cDNA is ready for:

• Blunt-end ligation into cloning vectors

• Sticky-end cloning using adapter ligation

• Restriction digestion using FastDigest enzymes

• Quantification via Nanodrop (A260) or gel visualization

Tips to Prevent RNase Contamination

• Use RNase-free tubes, tips, and reagents

• Always wear gloves and change them often

• Keep tubes sealed during reactions

• Use RNase inhibitors provided in the kit

For a verbal walkthrough of this process, check out my YouTube video embedded below.

🗣️ Join the Discussion!

What type of RNA are you converting to cDNA—mRNA or total RNA? And what cloning project are you working on?
Let’s chat in the comments below!

July 18, 2025 Tags : cDNA for cloning , cDNA synthesis , ds cDNA kit , Maxima H Minus , molecular cloning , RNA to cDNA

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