Standard Operating Procedure

 

CDNA Preparation

 

Hazards:

 

 

Protection:

Wear gloves

 

Waste:

 

Spill Clean-Up:

 

Procedure:

cDNA Preparation

Notes:

• Maximum of 5 µg RNA can be used per reaction
• Use Invitrogen Kit: SuperScript First-Strand Synthesis System for RT-PCR (in -20 Freezer)
• Do two of each à 1 of each is a no RT control

 

First strand reaction

            RNA/Primers Mixture

RNA                                         n µl                                         

Oligo(dT)_12-18 (0.5 mg/ml)            1 µl                                         

10mM dNTP mix                       1 µl                                         

DEPC H₂0                                to 10 µl

                                                                       

• 65ºC, for 5 min, place on ice for at least 1 min.
• Prepare the following reaction mixture, adding each component in the indicated order. (For n samples + 1 minus RT control, prepare the reaction mix for n + 2 reactions):

      Each Reaction

10X RT buffer                                                  2 µl

25 mM MgCl₂                                                    4 µl

0.1 M DTT                                                        2 µl

RNaseOUT Recombinant RNase Inhibitor         1 µl

 

• Add 9 µl of reaction mixture to each RNA/primer mixture, mix gently, and collect by brief centrifugation.
• Incubate at 42°C for 2 minutes.
• Add 1 µl (50 units) of SuperScript II RT to appropriate tubes (not to no RT!), mix, and incubate at 42° for 50 minutes.
• Terminate reactions at 70°C for 15 minutes. Chill on ice.
• Collect the reactions by brief centrifugation. Add 1 µl of RNase H to each tube and incubate for 20 minutes at 37°.