User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions
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'''RNA Extraction''' | '''RNA Extraction''' | ||
* Use RNeasy Mini Kit | * Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit. | ||
Samples (TRIzol-lysed U2OS samples from -80°C freezer) | Samples (TRIzol-lysed U2OS samples from -80°C freezer) | ||
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# Tube 4-A (mock) | # Tube 4-A (mock) | ||
Procedure notes (based on Carly's notes from [http://openwetware.org/wiki/Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2/2013/01/07 1/10/13]) | Procedure notes (based on Carly's notes from [http://openwetware.org/wiki/Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2/2013/01/07 1/10/13])<br> | ||
'''TRIzol steps''' | |||
* Clean all work surfaces and pipettors with RNase-zap. | |||
* Thaw TRIzol-lysed cells at room temp | * Thaw TRIzol-lysed cells at room temp | ||
* Under fume hood: Add '''100 μL chloroform''' to each sample | * 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. | * 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 | * Centrifuge in cold room for 15 min @ 12,000G | ||
* Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE) | * 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. | * Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. Mix and incubate at room temp (refer to RNeasy protocol) | ||
* | '''Spin-column steps''' | ||
* Spin tubes at top speed for 30 sec | * Open up one pink, sealed spin-column (with attached cap) per sample, label it, and place into collection tube(s). | ||
* Discard | * Transfer RNA/ethanol mix to each spin column. | ||
* Spin tubes at top speed for 30 sec | * Spin tubes at top speed for 30 sec (room temp). | ||
* Put collection vial in a fresh | * Discard flow-through liquid and add 700μL of RW1 Buffer to the spin column. | ||
* Add 500μL of RBE Buffer | * Spin tubes at top speed for 30 sec (room temp). | ||
* Spin tubes at top speed for 30 sec then discard | * Put collection vial in a fresh collection tube and discard the old one. | ||
* Repeat the above two steps again | * --> Add 500μL of RBE Buffer to the spin column. | ||
* Discard | * Spin tubes at top speed for 30 sec (room temp) then discard the liquid flow-through. | ||
* Transfer liquid to a fresh tube and add | * Repeat the above two steps again. --> | ||
* Let tubes sit for 1 min | * Discard the flow-through and spin again. | ||
* Spin tubes for 1 min | * Transfer liquid to a fresh 1.5 mL tube (RNase-free) and add 30 μL of RNase-free H<sub>2</sub>O. | ||
* Store at -80°C | * Let tubes sit for 1 min at room temp. | ||
* Spin tubes for 1 min (room temp). | |||
* Store at -80°C or proceed to RNA concentration measurement. | |||
Revision as of 13:31, 4 February 2014
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01/27/14
RNA Extraction
Samples (TRIzol-lysed U2OS samples from -80°C freezer)
Procedure notes (based on Carly's notes from 1/10/13)
Spin-column steps
cDNA Synthesis
Procedure notes
qRT-PCR - Plate K562_1/ U2OS
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