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

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'''RNA extraction'''
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'''New RNA extraction'''
* Follow the TRIzol/ RNeasy protocol
* Follow the TRIzol/ RNeasy protocol
* Use Carly's TRIzol samples K562 1, 2, 3, and 4
* Use Carly's TRIzol samples K562 1, 2, 3, and 4
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| <u>Sample</u> || <u>OD 260</u> || <u>260/280</u> || <u>ng/μL</u> || <u>Vol. to use for cDNA synth.</u>
| <u>Sample</u> || <u>OD 260</u> || <u>260/280</u> || <u>ng/μL</u> || <u>Vol. to use for cDNA synth.</u>
|-
|-
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| 1. K562 1     || 0.975 || 2.017 || 780.25 || 3.2 μL (2.5 μg)
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| 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 2  || 0.890 || 2.021 || 711.81 || 3.5 μL (2.5 μg)
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| 2. K562 4  || 0.696 || 2.020 || 556.52 || 4.5 μL (2.5 μg)
| 2. K562 4  || 0.696 || 2.020 || 556.52 || 4.5 μL (2.5 μg)
|}
|}
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* Conclusion: Success! Yields are very good. Continue with cDNA synthesis, 3 reactions per RNA.
'''oligo(dT) Primer annealing'''
'''oligo(dT) Primer annealing'''
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Revision as of 22:36, 4 June 2014

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

  • cDNA synthesis: K562 sets ## - ##

cDNA synthesis: K562

  • Use Carly's RNA from -80 °C
  • Assume that K562 1.1 is +PcTF and K562 2.1 is control (will find out 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 = 4
  • Samples:
  1. K562 1.1 (E004)
  2. K562 1.1 (E005)
  3. K562 2.1 (C004)
  4. K562 2.1 (C005)
Reagent Single rxn. Mix (x4)
10x RT buffer 2.0 8.0
25 mM MgCl2 4.0 16.0
0.1 M DDT 2.0 8.0
RNaseOUT 1.0 4.0
SuperScript III RT 1.0 4.0
  10.0 μL 40.0 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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