User:Karmella Haynes/Notebook/miR Trigger/2011/06/01

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06/01/11

  • ✓ Flow cytometry: miR sensor transfections
  • ✓ Transfections: new miR sensor constructs into JDS33-47



Flow cytometry
> Experiment/folder: Karmella mCherry AmCyan > 060111 MS Repression > Use 96-well round-bottom plate

  • Trypsinize cells w/ 0.5 mL trypsin medium
  • Harvest using 1 mL plain medium, transfer 1.5 mL cells to 15 mL conical
  • Spin at 1100 rpm @ 4°C
  • Pour off sup. and resuspend cells in 200 μL 1x PBS, transfer 200 μL to 96-well plate
  1. Specimen_001, wells A1-A3: blank
  2. Specimen_002, wells A4-A6: FC1
  3. Specimen_003, wells A7-A9: FC2
  4. Specimen_004, wells A10-A12: FC3
  5. Specimen_005, wells B1-B3: FC4
  6. Specimen_006, wells B4-B6: FC31
  7. Specimen_007, wells B7-B9: FC32
  8. Specimen_008, wells B10-B12: FC33
  9. Specimen_009, wells C1-C3: FC34
  10. Specimen_010, wells C4-C6: KAH100
  11. Specimen_011, wells C7-C9: KAH101
  12. Specimen_012, wells C10-C12: KAH118
  13. Specimen_013, wells D1-D3: KAH119
  14. Specimen_014, wells D4-D6: KAH121
  15. Specimen_015, wells D7-D9: KAH122
  16. Specimen_016, wells D10-D12: KAH124
  17. Specimen_017, wells E1-E3: KAH125


--> Results: Mostly no signal except for CFP from KAH100 and 101 (CFP-Reporter-only constructs)
--> Stop testing these constructs and re-design (See BioBrick Cloning entry 5/31/11)



Transfections
Plate 1: Lipo LTX
> Lipofectamine LTX > Follow Jason's protocol, 12-well plates

Plate 2: X-tremeGENE 9
> Got free sample from Roche. Try it out.

Wells Plasmid DNA Volume PLUS reagent Lipo Opti-MEM (total)
1, 2 KAH183/176-MV8 1 μg 5 μL 1 uL 3 μL 200 μL
3, 4 KAH184/176-MV8 " 5 μL " " "
5, 6 KAH183/177-MV8 " 5 μL " " "
7, 8 KAH184/177-MV8 " 5 μL " " "

--> 9, 10. no transfection

Lipo LTX
> Add 180 μL Opti-MEM to 5 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.
> Add 3 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

X-tremeGENE 9
> Add 3 μL reagent to 100 μL Opti-MEM, mix gently
> Add DNA --> R.T/ 30 min.
> Add ~100 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

> Add 1 μg/mL dox to second well of each sample pair