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:
Reagent
|
Volume
|
PowerUp
SYBR
|
5
uL
|
10uM
primer (F+R)
|
1
uL
|
ddH2O
|
1.5
uL
|
cDNA
|
2.5
uL
|
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.
声明:该视频作品仅代表作者观点,用于共享科学技术。内容仅供大家参考,不代表本站立场。