User:Karmella Haynes/Notebook/Polycomb project/2011/08/24

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08/24/11

  • ✓ Transfections: H3me Gluc-reporter
  • ✓ RNA prep/ cDNA: ES cell pellets from Manching Ku



Transfections: H3me Gluc-reporter stables
> See 8/12/11 (H3 Cyan-reporter stables)
> Lipofectamine LTX; follow Jason's protocol, 12-well plates
> Use ~1:3 ratio of FlpE plasmid to reporter plasmid
> Two different types of Flp-in T-REx: HEK293, U2OS (plated at a ~1:5 dilution, 1 mL per well yesterday)

Plate 1: HEK293 FTRx
Plate 2: U2OS FTRx

Wells Reporter plasmid (Gluc) 750 ng DNA = Recombinase 250 ng DNA =
1, 2. 234/V0200 (Gluc) 2.7 μL FlpE 0.5 μL
3, 4. 235/V0200 (DPRE-Gluc) 2.4 μL FlpE 0.5 μL


Lipo LTX: Make 4x reactions (2 wells per plate for each transgene)
> Dilute 1 μg DNA in dH2O (10 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA
> Add 1.0 μL PLUS reagent --> R.T/ 5 min.
> Add 3.0 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



RNA prep/ cDNA
> Transfected ES cell samples from Manching Ku; cells were pelleted and kept frozen at -80°C; cell line/ vector allows dox-inducible expression of transgene

  1. KAH160/pSB31 -dox
  2. KAH160/pSB31 +dox
  3. KAH165/pSB31 -dox
  4. KAH165/pSB31 +dox
  5. KAH170/pSB31 -dox
  6. KAH170/pSB31 +dox

> RNA prep
--> Used TriZol and chloroform to extract RNA (Note: pellets appeared to only partially lyse in TriZol solution after extended incubation and vortexing)
--> Isolated RNA from aqueous phase (after 15 min. centrifugation step) using QIAGEN RNeasy kit
--> Eluted with 50 μL RNase-free water (added eluate back to column for second elution to maximize concentration).

Sample A260 260/280 ng/μL 500 ng RNA =
1. 160 -dox 2.242 2.00 89.7 5.6 μL
2. 160 +dox 1.775 1.93 71.0 7.0 μL
3. 165 -dox 1.110 1.90 44.4 8.0 μL (355.2 ng)
4. 165 +dox 5.492 2.02 219.7 2.3 μL
5. 170 -dox 3.610 2.01 144.4 3.5 μL
6. 170 +dox 4.056 2.02 162.3 3.0 μL


> cDNA
--> Use Superscript III kit; 1 rxn per sample
--> Store remainder of RNA at -80°C


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