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

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06/01/11

 * &#x2713; Flow cytometry: miR sensor transfections
 * &#x2713; 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.

--> 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&deg;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&deg;C

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


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