Mike Barnkob:Protocols/Cloning/cDNA library from mRNA

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Creating cDNA from mRNA

Protocol on how to convert mRNA into cDNA.

General notes

Lab practice:

• Wear a clean lab coat.
• Change gloves vigorously.
• Use dedicated (or at least very clean) pipets.
• Clean lab bench (it probably needs it too).

Experimental setup:

• Use two types on negative control for experiment:
  1. One negative consisting of a duplicate sample but without the RT enzyme added.
  2. One negative consisting of the RT enzyme and master mix but with nuclease free water, instead of mRNA (so no mRNA in this sample).
• Use 500-1000ng RNA for each sample.

Reagents

Reagents:

• Applied Biosystems High Capacity cDNA Reverse Transcription Kit.
• Autoclaved 1.5 mL Eppendorf tubes.
• Thermal cycler
• Centrifuge with 96-well plate insert
• 96 well PCR plate and adhesive film
• Nuclease free dH20

2x RT master mix:
Keep all reagents on ice, including mastermix.

1. Allow reagents to thaw on ice.
2. For each sample (+ two controls), mix the follow:
   • 2 μL 10✕ RT Buffer
   • 0.8 μL 25✕ dNTP Mix
   • 2.0 μL 10✕ RT Random Primers
   • 1.0 μL RNase Inhibitor
   • 3.2 μL Nuclease-free dH2O
   • 1.0 μL MultiScribe Reverse Transcriptase
     Remember: Do not add this to the RT-negative control. Use 1.0 μL dH20 instead.
3. Mix gently by pipeting.

Protocol

Mix:

1. Pipette 10 μL of 2✕ RT master mix into each well of a 96-well reaction plate or individual tubes.
2. Pipette appropriate μL of RNA sample into each well (aiming for around 1000 ng pr. sample)
   Remember: Do not add mRNA to the mRNA-negative control. Use 1.0 μL dH20 instead.
3. Top up to a total of 10 μL with nuclease-free dH20. Pipet up and down to mix. Total sample size should now be 20 μL.
4. Seal the plates or tubes.
5. Centrifuge the plate at 500 RPM for 1 minute to eliminate any air bubbles.
6. Place the plate or tubes on ice until you are ready to load the thermal cycler.

Thermal cycler:

1. Program thermal cycler as follows:
   • Step 1: 25 °C for 10 min
   • Step 2: 37 °C for 120 min
   • Step 3: 85 °C for 5 min
   • Step 4: 4 °C for ∞
2. Load plate or tubes into cycler and run.

Storage:

1. Transfer to 1.5 mL Eppendorf tube, label and store at -20 °C.

Quality control:

1. ?

References

• Applied Biosystems High Capacity cDNA Reverse Transcription Kit Protocol, http://docs.appliedbiosystems.com/pebiodocs/04375575.pdf
• cDNA Synthesis Protocol from Toni Vidal-Puig's lab: