User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions
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* Use RNeasy Mini Kit | * Use RNeasy Mini Kit | ||
Samples ( | Samples (TRIzol-lysed U2OS samples from -80°C freezer) | ||
# Tube 1-A (+PcTF) | # Tube 1-A (+PcTF) | ||
# Tube 1-A (+PcTF) | # Tube 1-A (+PcTF) | ||
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Procedure notes | Procedure notes | ||
* | * Open up pink, sealed spin-columns | ||
* Thaw TRIzol-lysed cells at room temp | |||
* Under fume hood: Add '''100 μL chloroform''' to each sample | |||
* Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min. | |||
* Centrifuge in cold room for 15 min @ 12,000G | |||
* Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE) | |||
* Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. | |||
* Transfer to collection tube (we have K562 1, K562 2, SK-N-SH 1, and SK-N-SH 2) | |||
* Spin tubes at top speed for 30 sec | |||
* Discard collected liquid and add 700μL of RW1 Buffer | |||
* Spin tubes at top speed for 30 sec | |||
* Put collection vial in a fresh collector tube and discard of the old one | |||
* Add 500μL of RBE Buffer | |||
* Spin tubes at top speed for 30 sec then discard of the liquid waste | |||
* Repeat the above two steps again | |||
* Discard of the waste and spin again | |||
* Transfer liquid to a fresh tube and add 30mL of RNase-Free H20 | |||
* Let tubes sit for 1 min | |||
* Spin tubes for 1 min | |||
* Store at -80°C | |||
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Procedure notes | Procedure notes | ||
* Kit components kept on ice: RNase H, SS III RT, and RNase out | |||
* Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | |||
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Revision as of 17:33, 3 February 2014
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01/27/14
RNA Extraction
Samples (TRIzol-lysed U2OS samples from -80°C freezer)
Procedure notes
Procedure notes
qRT-PCR - Plate K562_1/ U2OS
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