Standard Protocol for a 12 well plate:

1. wash the cells with PBS
2. add 0.5 ml Schneider-Medium without FCS and without Glutamin to each well
3. add 15-30 µg dsRNA to each well, gently swirl the plate
4. incubate for 30-60 min at RT
5. add 2 ml Schneider's medium with FCS and glutamin to each well
6. incubate for three days (or longer, depending on the gene to be depleted)
7. two hours prior to fixation suspend the cells by pipetting the cells up and down. Add the coated cover slide to the well and let the cells adhere.
8. check whether the cells adhere. If yes, proceed with fixation for 20 min.

dsRNA synthesis

• Prepare a transcription template by PCR in 100 ul final volume (distribute to two vials).
  1 ug genomic DNA (or other template),
  1 ul primer1 (100 uM,1 uM),
  1 ul primer2 (100 uM, 1 uM),
  2 ul dNTP mix (50x, 0.2 mM final),
  10 ul 10x PCR Taq buffer (with Mg2+),
  4 U Taq polymerase.
• Perform a PCR reaction with 30-35 cycles. Optimise the protocol if necessary.
• Isolate the PCR product by agarose gel electrophoresis and Qiaquick. The yield should be about 5 ug (binding capacity of the columns)
• Set up an in vitro RNA transcription reaction with T7 RNA polymerase 50 ul final volume:
  1 ug DNA template with T7 sites,
  7.5 uM NTP (each),
  1x T7 buffer,
  2.5 u pyrophosphatase,
  4 u T7 RNA polymerase.
• Incubate at 37¡C for four hours.
• Purify the dsRNA by Phenol/chloroform extraction and ethanol precipitation. Dissolve in 50 ul water (DEPC treated).
• Measure the concentration. Analyse an aliquot by agarose electrophoresis. The yield should be about 250 ug.