Rna

Methanol/Chloroform extraction of whole cell small molecular weight metabolites from cells in suspension.

Alternative fixation procedure for FACS fixation, using formaldehyde fixatice.

This is an alternative to poly-L-lysine treatment. The APES coating makes the slides more adhesive for fixed tissue and living cells.

Titration of retrovirus lines. Note, target cells must be dividing to be infected. As the viral RNA/protein complex cannot enter the nucleus entry must wait for nuclear membrane to dissolve at mitosis.

Retroviral infection of primary human keratinocytes (PHKs) # PHK Key Points PHKs must be actively dividing to be successfully infected. It is important not to allow cells to become confluent as this will prevent trypsinization (even if you do force them all off only 10% will re-attach in the new flask). PHKs must be allowed to 'get going' after resuscitation from freezing, try to infect at the 4–16 cell cluster size (3–4 days?). PHKs differentiate in response to serum and/or Ca++. Apparently they are able to de-differentiate if the exposure was not too long/strong. # Packaging cell points to bear in mind Packaging cells grow very fast especially PT67s (12-hour doubling time). Overconfluence is not so bad (titres remain OK) Half-life of virus is about 4 hours in media at 37ºC. Supernatants are useful at anything from 6 hours to 24 hours. More than this then defective particles may become a significant factor although at our low titres it is not such an issue.

Lacritin Stimulated Cell Proliferation Assay without siRNA Knockdown or Inhibitors

Lacritin Stimulated Cell Proliferation Assay with siRNA or Inhibitors

Isolation of RNA from whole worms using TRIZOL reagent (Gibco-BRL)

Protocol for bacterial mediated RNAi