User:Karmella Haynes/Notebook/miR Trigger/2009/12/22

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

12/22/09

  • ✓ Minipreps: KAH93-95/MV2 (2 each)
  • ✓ Transfection 1: KAH93, 94, 95/MV2 for microscopy and RT-PCR (6-well, Lipo)
  • ✓ Transfection 2: KAH94, 95/MV2 for miRNA dose response curve (24-well, Fugene)
  • ✓ Re-transform plasmids: KAH93, 94, 95/MV2 (Did not work well, re-try with Z-DH5α, 12/23/09 ✓)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH93/MV2 = 4839
3,4. KAH94/MV2 = 5011
3,4. KAH95/MV2 = 5083
E/P digest 12/22/09
15 μL/lane; 1% agarose
DNA(plasmid) 2.0
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

> No plasmid DNA. Re-transform from minipreps (quick & dirty transformation, DH5α T1R lab stock)


Transfection 1
> Details

Wells Sample DNA Volume Lipo Opti-MEM (total)
1 KAH93/MV2 2 μg 4.4 μL 4 μL 500 μL
2 KAH93/MV2(Dox) " 4.4 μL " "
3 KAH94/MV2 " 4.8 μL " "
4 KAH94/MV2(Dox) " 4.8 μL " "
5 KAH95/MV2 " 8.7 μL " "
6 KAH95/MV2(Dox) " 8.7 μL " "
7 KAH108/MV2 " 4.6 μL " "
8 KAH108/MV2(Dox) " 4.6 μL " "
9 mock --- --- " "
10 mock(Dox) --- --- " "
11 untreated --- --- --- "
12 untreated(Dox) --- --- --- "

> Add 2x Lipo to 500 μL Opti-MEM --> R.T/ 5 min.
> Add 2x DNA to 500 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml total vol.); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)

Day 2
> Add 1μg/mL Dox to Dox+ samples. Incubate overnight (~20 hours).

Day 3
> RNA preps/ cDNA synthesis


Transfection 2
> Details

Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH94/MV2 150 ng 0.4 μL 1.8 μL 18.2 μL
7-12 KAH95/MV2 " 1.2 μL " "
13-18 KAH108/MV2(ctrl) " 0.3 μL " "
19-24 mock " --- " "

> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min.
> Add 6x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C overnight

Day 2
> Refresh w/ ab-free, no phenol red medium
> Add varying doses of doxycycline to the wells in each row: 0, 0.1, 1.0, 10.0, 100.0, 1000.0 ng/mL
---> 1:10 serial dilutions; dilute dox in 50 μL medium
> Assay RFP/YFP after 5 or 6 hours (plate reader)
--> Accidentally did 1:100 serial dilution (0, 0.00001, 0.001, 0.1, 10.0, 1000.0) for Read #1
--> Refreshed medium and used correct 1:10 dilutions of Dox for overnight induction > Grow overnight

Day 3
> Read #2: Assay RFP/YFP (morning)



Personal tools