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
- Specimen_001, wells A1-A3: blank
- Specimen_002, wells A4-A6: FC1
- Specimen_003, wells A7-A9: FC2
- Specimen_004, wells A10-A12: FC3
- Specimen_005, wells B1-B3: FC4
- Specimen_006, wells B4-B6: FC31
- Specimen_007, wells B7-B9: FC32
- Specimen_008, wells B10-B12: FC33
- Specimen_009, wells C1-C3: FC34
- Specimen_010, wells C4-C6: KAH100
- Specimen_011, wells C7-C9: KAH101
- Specimen_012, wells C10-C12: KAH118
- Specimen_013, wells D1-D3: KAH119
- Specimen_014, wells D4-D6: KAH121
- Specimen_015, wells D7-D9: KAH122
- Specimen_016, wells D10-D12: KAH124
- 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
|
Project name
Main project page
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