Bioanalytical Research Group

iTAP Based Protein Complex Purification

RNA interference protocol

We use the Promega Ribomax Large Scale RNA Production System T7 (Cat. No.: P1300) to produce dsRNAs and the Jack Dixon protocol (downloaded version,15.1.2003) to prepare dsRNAs.

I. Preparation of Template DNA:

1. Design two oligos for your gene of interest. Each should incorporate a 5' T7 RNA polymerase binding site, resulting in a PCR product of approximately 700 bp.

2. T7 RNA polymerase binding site: TTA ATA CGA CTC ACT ATA GGG AGG

3. Purify the PCR DNA template It should be free of RNAses and inhibitors such as high salt, detergents or EDTA.

4. Quantify the PCR Product on an agarose gel. The dsRNA reaction detailed below requires 5-10 µg total DNA in 40 µl.

II. Preparation of the dsRNA We use the Promega Ribomax Large Scale RNA Production System T7. We modify the supplied protocol as follows:

1. Prepare the templates as described above.

2. Add the following reagents from the kit in a 1.5 ml Eppendorf tube at RT. For a 100µl reaction:

 20 µl Buffer 5x

 30 µl rNTPs (25 µM)

 DNA 5 µg

 10 µl T7 enzyme mix

 nuclease free water to a final volume of 100 µl

3. Mix tube gently, spin contents down and incubate at 37˚C for 2 to 6 hours or overnight.

4. Add 2 µl of RNAse free DNAse. Incubate for 1 h at 37¾C. Add 200 µl of nuclease free water and extract the RNA with Phenol:Chloroform

5. Ethanol precipitate your reactions

6. Resuspend the pellet in 100 µl - 200 µl water.

7. Heat your reactions tubes at 70˚C for 30 min.

8. Place tubes in a beaker of 80˚C water on the bench and slowly cool to room temperature to anneal the RNA.

9. Adjust the final concentration of your dsRNA to 3 µg/µl, using the following calculation: OD260nm 1 = 45 µg/ml

10. Run 1-2 µg of your dsRNA on a 1% Agarose gel to check the integrity and size of the dsRNA. A 100 µl reaction typically yields up to 1 mg of dsRNA.