User:Karmella Haynes/Notebook/PcTF Genomics/2014/06/04

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(06/04/14)
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Pc-TF Genomics Main project page
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06/04/14

  • cDNA synthesis: K562 sets 4 - 9



cDNA synthesis: K562

  • Use Carly's RNA from -80 °C
  • K562 1 and 3 are +PcTF and K562 2 and 4 are controls (see 12/18/12 ...will confirm when QC plate for mCh RT-PCR is run)
  • Will hold off on continuing U2OS for transiently transfected samples; will switch to KAH126 for further analyses


Measure RNA concentration

Sample OD 260 260/280 ng/μL Vol. to use for cDNA synth.
1. K562 1.1 -0.006 1.968 -5.193 n/a
2. K562 2.1 -0.003 1.6 -2.724 n/a
  • Conclusion: discard samples
  • Do a new RNA extraction from TRIzol samples


New RNA extraction

  • Follow the TRIzol/ RNeasy protocol
  • Use Carly's TRIzol samples K562 1, 2, 3, and 4


Measure RNA concentration

Sample OD 260 260/280 ng/μL Vol. to use for cDNA synth.
1. K562 1 0.975 2.017 780.25 3.2 μL (2.5 μg)
2. K562 2 0.890 2.021 711.81 3.5 μL (2.5 μg)
2. K562 3 0.856 2.028 684.53 3.7 μL (2.5 μg)
2. K562 4 0.696 2.020 556.52 4.5 μL (2.5 μg)
  • Conclusion: Success! Yields are very good. Continue with cDNA synthesis, 3 reactions per RNA.


oligo(dT) Primer annealing

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50 μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
DEPC-treated water = 8.0 - vol. total RNA
  10.0 μL

--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.


cDNA synthesis mix

  • Total reactions = 12
  • Samples:
  1. K562 1 (E004)
  2. K562 1 (E005)
  3. K562 1 (E006)
  4. K562 2 (C004)
  5. K562 2 (C005)
  6. K562 2 (C006)
  7. K562 3 (E007)
  8. K562 3 (E008)
  9. K562 3 (E009)
  10. K562 3 (C007)
  11. K562 3 (C008)
  12. K562 3 (C009)
Reagent Single rxn. Mix (x12)
10x RT buffer 2.0 24.0
25 mM MgCl2 4.0 48.0
0.1 M DDT 2.0 24.0
RNaseOUT 1.0 12.0
SuperScript III RT 1.0 12.0
  10.0 μL 120.0 μL

--> Aliquot 10 μL of mix into 8-tube strip
--> Add annealing rxn. into each 10 μL aliquot
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.
--> Store at -20°C





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