Oct 17, 2022
Real time qPCR (Fly Heads)
- Mel Feany1,2
- 1Brigham and Women's Hospital;
- 2Harvard Medical School
- Daniel's workspace
Protocol Citation: Mel Feany 2022. Real time qPCR (Fly Heads). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y4r1gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71455
Keywords: ASAPCRN, rt qpcr with rna, rt qpcr, fly head, real time, rna, protocol
Abstract
This Protocol describes how to perform RT qPCR with RNA extracted from fly heads.
RNA Extraction
Homogenize 6 heads in 50 µL Qiazol, then add 450 µL Qiazol.
Spin 00:10:00 at 4 °C at 12,000 x g.
10m
Incubate supernatant for 00:05:00 at Room temperature .
5m
Add 100 µL chloroform. Vortex, incubate for 00:03:00 at Room temperature , then spin for 00:15:00 at 4 °C at 12,000 x g.
18m
Transfer the upper phase to fresh tube, add 250 µL isopropanol, incubate 00:10:00 at Room temperature , spin for 00:10:00 at 4 °C at 12,000 x g.
20m
Wash the pellet in 500 µL 75% ethanol (it will be very loose) three times. Do not vortex.
Air dry
Resuspend in 15 µL RNAse/DNAse-free water.
Measure 260/280 and 260/230 on nanodrop.
RT-PCR
Use 1 µg RNA to perform RT-PCR reaction.
Thermocycler settings
a. 25 °C 00:10:00
b. 37 °C 02:00:00
c. 85 °C 00:05:00
d. 4 °C Hold
2h 15m
qPCR
Make master mix with primers, SYBR Green and water.
Dilute DNA 1:50 making a 50 µL stock.
Add 2 µL DNA to wells with special tips.
Add 14 µL master mix to wells with regular tips. Pipette up and down once but do not expel a bubble. Keep master mix on ice the entire time to avoid bubbles.
Check for bubbles and run on machine.