Oct 17, 2022

Public workspaceReal time qPCR (Fly Heads)

DOI
dx.doi.org/10.17504/protocols.io.q26g7y4r1gwz/v1
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School
Daniel El Kodsi
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DOI: dx.doi.org/10.17504/protocols.io.q26g7y4r1gwz/v1
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
Abstract
This Protocol describes how to perform RT qPCR with RNA extracted from fly heads.
RNA Extraction
RNA Extraction
Homogenize 6 heads in Amount50 µL Qiazol, then add Amount450 µL Qiazol.

Spin Duration00:10:00 at Temperature4 °C at 12,000 x g.

10m
Incubate supernatant for Duration00:05:00 at TemperatureRoom temperature .

5m
Add Amount100 µL chloroform.  Vortex, incubate for Duration00:03:00 at TemperatureRoom temperature , then spin for Duration00:15:00 at Temperature4 °C at 12,000 x g.

18m
Transfer the upper phase to fresh tube, add Amount250 µL isopropanol, incubate Duration00:10:00 at TemperatureRoom temperature , spin for Duration00:10:00 at Temperature4 °C at 12,000 x g.

20m
Wash the pellet in Amount500 µL 75% ethanol (it will be very loose) three times.  Do not vortex.

Air dry
Resuspend in Amount15 µL RNAse/DNAse-free water.

Measure 260/280 and 260/230 on nanodrop.
RT-PCR
RT-PCR
Use Amount1 µg RNA to perform RT-PCR reaction.

Thermocycler settings
a.     Temperature25 °C Duration00:10:00
b.     Temperature37 °C Duration02:00:00
c.      Temperature85 °C Duration00:05:00
d.     Temperature4 °C Hold

2h 15m
qPCR
qPCR
Make master mix with primers, SYBR Green and water.
Dilute DNA 1:50 making a Amount50 µL stock.

Add Amount2 µL DNA to wells with special tips.

Add Amount14 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.