User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions
From OpenWetWare
Line 30: | Line 30: | ||
* 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 (4°C) 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. Mix and incubate at room temp (refer to RNeasy protocol) | * 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) | ||
Line 47: | Line 47: | ||
* Let tubes sit for 1 min at room temp. | * Let tubes sit for 1 min at room temp. | ||
* Spin tubes for 1 min (room temp). | * Spin tubes for 1 min (room temp). | ||
'''Measure RNA Concentration''' | |||
* Use the BioTek Take3 plate/ reader | |||
{| | |||
| Sample || OD 260 || 260/280 || ng/μL | |||
|- | |||
| 1. 1-A (+PcTF) || 1.007 || 2.1 || 805.8 | |||
|- | |||
| 2. 1-A (+PcTF) || 0.843 || 2.1 || 674.0 | |||
|- | |||
| 3. 4-A (+PcTF) || 0.992 || 2.1 || 793.9 | |||
|- | |||
| 4. 4-A (+PcTF) || 0.959 || 2.1 || 767.0 | |||
|} | |||
* Keep on ice. Store stock RNA at -80°C after samples are used for cDNA synthesis. | |||
Line 57: | Line 70: | ||
'''cDNA Synthesis''' | '''cDNA Synthesis''' | ||
* Use Superscript III kit (freezer) | * Use Superscript III kit (freezer) | ||
* Pre-heat the heat block to 65°C. Use the PCR strip-tube-compatible block | |||
Procedure notes | Procedure notes | ||
* Kit components kept on ice: RNase H, SS III RT, and RNase out | * 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 | * Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | ||
* Label four 0.2 mL PCR strip-tubes | |||
* Make 8 μL solutions that contain 2.0 μg RNA | |||
** vol stock RNA (μL) = 2000 ng/ [RNA] from table | |||
** vol H<sub>2</sub>O = 8.0 μL - vol stock RNA | |||
* Make duplicates for each sample. Use RNA stocks #1 and #3. | |||
# Reaction 1: 2.5 μL Stock RNA #1 + 5.5 μL H<sub>2</sub>O = 2.5ug | |||
# Reaction 2: 2.5 μL Stock RNA #1 + 5.5 μL H<sub>2</sub>O = 2.5ug | |||
# Reaction 3: 2.5 μL Stock RNA #3 + 5.5 μL H<sub>2</sub>O = 2.5ug | |||
# Reaction 4: 2.5 μL Stock RNA #3 + 5.5 μL H<sub>2</sub>O = 2.5ug | |||
'''Part 1''' | |||
* Add 1μL of primer (Oligo dT) | |||
* Add 1μL of dNTP | |||
* Total volume is 10 μL | |||
* Incubate for 5 min @ 65°C | |||
* Immediately place on ice and incubate for 1 min. | |||
'''Part 2''' | |||
* Make a master mix for 4 rxns: | |||
** Single reaction = 2 μL RT Buffer, 4 μL MgCl2, 2 μL DTT, 1 μL RNaseOut, 1 μL SuperScript III | |||
** 4 reactions = 8 μL RT Buffer, 16 μL MgCl<sub>2</sub>, 8 μL DTT, 4 μL RNaseOut, and 4 μL SuperScript III | |||
* Carefully add 10μL of MM to each rxn tube | |||
* Mix tubes gently by flicking, centrifuge for a few seconds | |||
'''Part 3''' | |||
* RT-PCR Machine, set up as follows: | |||
# Stage 1: 50 min @ 50°C | |||
# Stage 2: 5 min @ 85°C | |||
# Stage 3: Infinity @ 4°C | |||
* Add 1μL: of RNase H | |||
* Incubate @ 37°C for 20 min | |||
* Store at -20°C | |||
---- | ---- |
Revision as of 13:56, 4 February 2014
Pc-TF Genomics | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
Part 1
Part 2
Part 3
qRT-PCR - Plate K562_1/ U2OS
|