qPCR - 384 well plate

一、视频摘要

RT-qPCR is used in a variety of applications including gene expression analysis, pathogen detection, genetic testing, and disease research. Here we show how to load cDNA samples into 384-well qPCR plate, which can be used for running more samples on one plate. Compared with 96-well plate, 384-well plate is really convenient and can save our time.

二、关键词

qPCR, 384-well plate, electronic pipette

三、实验样品信息，试剂、耗材或仪器

1. 样品信息

2. 试剂和耗材

Materials:

Filter tips (1mL, 200uL, 10uL)

Electronic pipette (10-100uL, 1-10uL)

Eppendorf pipette (1mL, 200uL)

RNase AWAY™ Surface Decontaminant Spray

Marker pen

Optical adhesive covers

Ice & ice block

75% Ethanol

PowerUp SYBR master mix

Primers

cDNA

3. 仪器和软件

Machine:

Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System, 384-well, laptop

Catalog number: A28570

Software:

Design and Analysis Software v1.5.2, QuantStudio 3 and 5 systems

Electronic pipette:

FP-Novus 1-10ul Micro Electronic pipette, 46200000

FP-Novus 10-100ul Electronic pipette, 46200400

四、实验操作

1. Spray 75% Ethanol and RNase away on your hands. (Note: Be careful that do not spay into the 384-well plate.)

2. Prepare Mix by adding SYBR mix, primer and ddH₂O according to the following program:

Note: Remember to vortex primers before adding (not shown in the video)

3. Gently vortex the prepared Mix and spin it down.

4. Using electronic pipette (choose the function: stepper) to load the Mix and cDNA into 384-well plate. Adjust the volume and speed before loading samples.

Note: Load the Mix on the left side of the well.

Then load the cDNA (Note: Load the cDNA into the center upper side of the well).

5. Gently shake the plate to shake the cDNA down.

6. Cover the optical adhesive covers carefully.

7. Centrifuge the plate shortly. Please note the orientation of the plate.

8. Carefully take the plate out. It's ready to be run in the machine.

五、注意事项

As shown in Procedure section.

六、结果分析（可选）

七、参考文献（可选）

Bustin S. (ed) (2004) A-Z of Quantitative PCR. IUL Biotechnology Series, International University Line, La Jolla, California.