RNA Extraction Protocol for use in the Clontech Creator SMART cDNA Library Construction Kit

JD Swanson

Huck Institute of Life Sciences

Penn State University

University Park

PA 16802

Foreword

This is a modification of the Ambion RNAqueous kit (Cat # 1912) in combination with the Plant RNA Isolation Aid (Cat # 9690). I designed this protocol out of a need for good quality cacao flower RNA for use in cDNA library construction. It is a very quick and easy protocol. It is designed to extract RNA in the range of 50-100ng/uL.

Don’t forget that this is for RNA, always wear gloved and always use RNA free tubes and tips, or DEPC treated lab ware. Most solutions are provided with the kit (except Ethanol). All you really need are tips, 100% ethanol, mortar and pestle, and small pestles, some 1.5mL tubes.

Protocol

1) Use 100uL of Plant RNA isolation mix with 800uL of lysis binding mix.

2) Collect 100mg of tissue and quickly Freeze tissue in liquid nitrogen.

a. Grind with DEPC treated mortar and pestle

b. Add 450uL buffer prepared in 1) to half of the ground tissue in a 1.5mL tube

c. Repeat with the other half

d. Mash tissue further with small pestle

3) Spin for 5 min at 10,000 x g, while spinning get Elution solution and preheat to 70^oC

4) Transfer supernatant to a new 1.5mL tube

5) Add an equal volume of 64% ethanol (~400uL)

6) Assemble filter cartridge and collection tube, add lysate from 5) to top of filter cartridge

7) Spin for 1 min at 10,000 x g, discard flow through. (The RNA is now on the filter)

8) Add 700uL of wash solution #1, spin for 1 min at 10,000 x g, discard flow through

9) Add 500uL of wash solution #2/3, spin 1 min at 10,000 x g, discard flow through

10) Repeat 9)

11) Spin 10-30 sec to remove the last traces of Wash solution from the column

12) Get a fresh collection tube

a. Add 40uL elution solution (preheated in step 3)) to centre of filter in column.

13) Spin for 30 sec at 10,000 x g. (The RNA is now in the collection tube)

14) Add a second aliquot (20uL) of elution solution to the centre of the filter in the column. Spin for 30 sec at 10,000 x g. Use the same collection tube as used in 12)

15) Mix RNA with 30 uL of LiCl precipitation solution

a. Incubate at -20^oC for at least 30 min

16) Spin for 15 min at top speed

17) Carefully remove supernatant with a pipette and wash in 70% cold ethanol

18) Air dry pellet (~10min)

19) Re dissolve pellet in the desired amount of DEPC water (I use 60uL)