Harvest of Transfection Plates
From CCGB
Harvest of Transfection Plates
--by Hao Wang
All manipulationscan be performed in non-sterile tubes with non-sterile pipets.
1. Remove plate contents to labeled 15 ml centrifuge tubes (caps not necessary).
2. Cntrifuge @ speed 4 for 4 min (IEC Clinical, ~ 300g).
3. Pout off supernatant and suspend pellet in ~ 3 ml PBS.
4. Centrifuge again to wash, same conditions.
5. Pour off supernatant, suspend in 1 ml PBS and transfer to labeled microcentrifuge tubes.
6. Centrifuge in microfuge @ 2000 rpm for 4 min (Eppendorf).
7. Completely remove supernatant by aspiration using a 200 μl pipet tip attached to aspirator hose (no need to change tips between samples).
8. Make n+2 volumes of lysis buffer, hold @ r.t., and save leftover buffer for reagent blanks during following assays.
9. Suspend pellets in 100 μl appropriate lysing buffer.
a. β-GAL (lacZ): use GalactoLight Lysis Buffer @ r.t. This buffer is stored in refrigerator in the GalactoLight Kit. Add 0.5 μl 1M DTT per ml to appropriate volume of buffer. DTT should be added fresh each time.
b. Luciferase: use Cell Culture Lysis Reagent, stored in white freezer. Dilute 5 × to 1 × with water.
c. Dual Luciferase (Renilla): use Dual Luciferase Passive Lysis Buffer, stored in white freezer. Dilute 5 × to 1 × with water.
10. Hold @ r.t. for 10 min.
11. Centrifuge @ full speed for 1 min in Eppendorf microfuge.
12. Use a toothpick to remove any remaining pellet.
13. Lysate can be used immediately for appropriate assay or frozen at -80°C until use.
Note: Always remember to control for cell number vagaries by running a protein assay on 2, 4, or 8 μl of lysate. Use BCA aaasy (Pierce) and same volume of lysis buffer for reagent blank on all assays.
