Dec 12, 2024
RT Quantitative PCR, 384 well format
Forked from Quantitative PCR, 384 well format
- 1Indiana University School of Medicine
Protocol Citation: Malu G Tansey 2024. RT Quantitative PCR, 384 well format. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqdn1ovk5/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: December 11, 2024
Last Modified: December 12, 2024
Protocol Integer ID: 115042
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020527
Abstract
Quantitative PCR, 384 well format
Materials
Materials:
- PCR 8-tube strips or 96 well plates depending on sample# and gene# (General stores 4868)
- PCR strip tube lids (General stores 1400-0800)
- ABI Sybr Green (ABI 4301955)
- QPCR plate (ABI 4309849)
- Adhesive plate film (ABI 4311971)
- Brown plastic adhesive helper
- Filtered 200 μl pipette tips (2 boxes for one complete plate)
- Electronic Multichannel Pipette (referred to as Hilda from here on out)
- Primers – final concentration of 1.25 μM each
- Autoclaved MilliQ H2O
Set-up:
Set-up:
Sample cDNA: thaw your cDNA on ice (vortex and quick spin before plating)
Primer stock (+/-): Add 10 µl of (+) and 10 µl (-) to 180 µL MilliQ H2O
PCR Master Mix (12ul rxn) per sample per gene:
| A | B | |
| MilliQ H2O | 2 ul | |
| SYBR Green | 6 ul | |
| primers (2.5 μM each set) | 2 ul | |
| Total master mix | 10 ul |
Equipment: ABI 7900 Prism
Create each master mix in 1.5 mL Eppendorf tube per gene of interest:
Ex. if n =12) Add 6 uL SYBR x 2 reps x (12+3) samples = 180 uL SYBR per gene; 2 uL H2O x 2 reps x 15 samples = 60 uL H2O, and it would be 60 uL primer pairs too.
Procedure:
Procedure:
Add 2 µl diluted cDNA (1:10 in DI H2O) to the bottom of each 384 well, use 10 uL multichannel pipette (keep on ice)
Dispense 10 µl of cDNA/Mastermix (use 10uL multichannel pipetor after pouring gene master mix into reservoir for easy multichannel access) into each well on 384 well plate according to plate layout made in advance (keep plate on ice)
Gently blot top of plate with kimwipe (to keep samples from transferring to other wells)
Place clear Adhesive plate cover over the plate.
use brown ‘helper’ to smooth out
pay attention to edges
work from center of the plate out
Spin plate for 5 min at 3500 rpm (4° C)
During spin: set up ABI SDS program (keep plate in centrifuge until ready to run)
Seal plate with sticky film. Vortex and spin down plate 3500rpm for 5 min at 4C
Open SDS 2.3 program
File -> new
One instrument tab: real time -> Connect to machine -> open/close door
Insert plate, aligning A1 to A1
Close door
On layout tab; highlight unused wells, click “omit wells”
Highlight used wells and click “add detector” for each specific gene
Set to 10uL Rxn VL
Check cycle times and temperatures
Add dissociation stage (SYBR primers only)
| A | B | C | D | E | F | G | H | |
| Temp C | 50 | 95 | 95 | 60 | 95 | 60 | 95 | |
| Time | 2:00 | 10:00 | 0:15 | 1:00 | 0:15 | 0:15 | 0:55 |
Stage C&D 40 time
Run plate
Primer Validation Procedure: Set-up is same as above plus cDNA standard curve for each gene in an extra set of 8-tube PCR strips (see workflow file)