SMARTer_RACE_5_3__Kit_User_Manual_022714(11)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

V. Generating RACE-Ready cDNA

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING

A. General Considerations

We recommend using the Tricine-EDTA Buffer provided in the kit to resuspend and dilute your

cDNA samples throughout the protocols in this user manual, because Tricine buffers maintain their

pH at high temperature better than Tris-based buffers. Tris-based buffers can lead to low pH

conditions that degrade DNA.

Resuspend pellets and mix reactions by gently pipetting the solution up and down or by flicking the

bottom of the tube. Always spin tubes briefly prior to opening to collect the contents at the bottom of

the tubes.

Perform all reactions on ice unless otherwise indicated.

Add enzymes to reaction mixtures last.

Ethidium bromide (EtBr) is a carcinogen. Use appropriate precautions when handling and disposing

of this reagent. For more information, see Molecular Cloning: A Laboratory Manual by Sambrook &

Russell (2001).

B. Preparation and Handling of Total and Poly A+ RNA

1. General Precautions

The integrity and purity of your total or poly A+ RNA starting material is an important element in

high-quality cDNA synthesis. The following precautions will help you avoid contamination and

degradation of your RNA:

Have a separate bench and/or pipette set dedicated to RNA work, free of RNase

contamination.

Wear gloves throughout to protect your RNA samples from nucleases.

Use freshly deionized (e.g., MilliQ-grade) H2O directly, without treatment with DEPC

(diethyl pyrocarbonate). Takara Bio also offers RNase-Free Water (Cat. No. 9012).

Use only single-use plastic pipettes and pipette tips. Filter tips are recommended.

RNA Isolation

Clontech® offers several kits for isolating total or poly A+ RNA from a variety of sources:

Purified Product Starting Material

Total RNA

mRNA Plant or fungal samples Total RNA derived from

cultured cells or animal tissues Product NucleoSpin RNA Plant Magnosphere UltraPure mRNA Purification Kit Cat. #

740949.50

9186 2.

Many procedures are available for the isolation of poly A+ RNA (Farrell, 1993; Sambrook et al.,

2001).

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