User:Karmella Haynes/Notebook/miR Trigger/2009/12/27

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12/27/09

  • ✓ Minipreps: KAH93, 94, 95/MV2 (from re-transformation colonies)
  • ✓ Transfections: KAH120-125/MV2 (12-well format, Fugene)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH93/MV2 = 4839
3,4. KAH94/MV2 = 5011
5,6. KAH95/MV2 = 5083
File:KAH 122709 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.

Fugene transfection
> KAH120-125/MV2 + U2OS plain (12-well format, Fugene)

Wells Plasmid DNA Volume Fugene Opti-MEM
1 KAH120/MV2 400 ng 2.5 μL 3.6 μL 46.4 μL
2 KAH121/MV2 " 5.3 μL " "
3 KAH122/MV2 " 4.8 μL " "
4 KAH123/MV2 " 4.0 μL " "
5 KAH124/MV2 " 2.3 μL " "
6 KAH125/MV2 " 5.0 μL " "
7 mock --- --- " "

> 3.6 μL Fugene + 46.4 μL Opti-MEM --> R.T/ 5 min.
> Add DNA --> R.T./ 20 min.
> Add 50 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight

Day 2
> Check RFP/CFP expression
--> Experiment looks interesting so far. Document images and repeat again with full set of 9 pairs when KAH117-119 are finished