FastLane Cell Multiplex Kit (200)
PCR Multiplex PCR - Mammalian DNA
Publication protocol
Monocyte-derived macrophages from each animal were seeded, in triplicates, in a 96-well plate. Each well was seeded with 100 µL cell suspension containing 40,000 cells in the growth medium. After incubation for 48 h, they were induced with 1 µg/ml lipoprotein Pam3CSK4 (Invivogen cat No. tlrl-pms). Cells in control wells were kept uninduced. Six h after induction, cells were processed for real-time, one-step RT-PCR using a FastLane Cell Multiplex Kit (Qiagen cat No. 216513) following the protocol recommended by the manufacturer. Briefly, media from the wells were aspirated and to each well 125 µL of Buffer FCW was added. Immediately after this, Buffer FCW was removed and to each well 50 µL of cell processing mix was added and the plate was incubated at room temperature for 5 min. The FastLane cell lysates containing stabilized RNA obtained in this way from each well were then transferred to 0.6 ml centrifuge tubes and incubated for 5 min at 75°C. The concentration of the RNA was determined using Qubit RNA Assay Kit (Invitrogen cat No. Q32852) and Qubit 2.0 Fluorometer (Invitrogen cat No. Q32866). Finally, the concentration of all the samples was adjusted to 20 ng/µL.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
5 years ago
Author:
R. Verma
Multiplex PCR and qPCR
I have been doing multiplex PCR and qPCR separately. Is there a way to combine them in a single reaction using Sybr Green?
Discussion
5 years ago
Author:
Roland Lafontaine
My multiplex PCR has decreased in sensitivity
Hello there! My multiplex PCR has decreased in sensitivity even though I have not changed any of the parameters. The materials are in good condition and the PCR machine is well calibrated. I have tested multiple samples but one of my targets has decreased in sensitivity.
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Manufacturer protocol
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