Doxorubicin labs
This lab uses Doxorubicin hydrochloride (Topoisomerase II inhibitor) treated vs untreated cells and looks at expression levels of p21 and GAPDH by RT PCR and WB.
- 1 = RLT (RNA) Doxo Treated
- 2 = RLT (RNA) Untreated
- 1P = RIPA (Protein) Doxo Treated
- 2P = RIPA ((Protein) Untreated
- For RNA, use 1x10^6 cells per 350ul sample
- Prepare 2.8x10^6 / ml and aliquot into 350ul
- For Protein, use 2x10^6 cells per 100ul sample
- Prepare 2x10^7 / ml and aliquot into 100ul
2023 Class Numbers as a rough guide for prep numbers:
5229 Lab
- Doxo + (RLT) = 60 samples [prepared 76]
- Doxo - (RLT) = 60 samples [prepared 75]
HSBS Lab
- Doxo + (RLT) = 80 samples [prepared 100]
- Doxo - (RLT) = 80 samples [prepared 100]
- Doxo + (Ripa) = 50 samples [prepared 62]
- Doxo - (Ripa) = 50 samples [prepared 62]
Notes on Cell harvesting:
- Remove 25ml media from T-150 flask
- Rinse flask with ~5ml PBS (-Ca, -Mg)
- Trypsinise with 5ml 1x Trypsin/EDTA (Sigma T3924)
- Incubate at room temperature until cells release (check under microscope for detachment...scrape if needed!)
- Inactivate trypsin by addition of 5ml DMEM*
- Pool flasks into 50ml falcon tubes
- Centrifuge at 300 x g for 3 min to pellet
- Remove liquid without disturbing the pellet
- Re-suspend pellet in 10ml PBS
- Count with countess (10ul Trypan blue + 10ul cells)
Harvest for RNA Experiments:
- Each sample = 1x10^6 cells in 350ul RLT buffer
- Re-suspend pellet in RLT to concentration of 2.8x10^6 cells/ml and split into 350ul aliquots
e.g. Cell count = 1.2x10^7 cells/ml
Want 2.5x10^6 cells/ml
1.2x10^7 / 2.5x10^6 = 4.8ml (13 x 350ul samples)
Harvest for Protein Experiments:
- Each sample = 2.5x10^6 cells in 100ul Ripa buffer (+P+P)
- Re-suspend pellet in Ripa
