IFT57 primers were designed on the RefSeq NM_018010 (Table S1 ). PCR fragments were purified with the multiscreen Vacuum Manifold system (Millipore). Sequencing was performed with the ABI BigDye Terminator Cycle Sequencing kit (v.3.1) (Applied Biosystems) in ABI 3130 sequencer 7 (Applied Biosystems) according to the manufacturer’s instructions. Sequence data were analyzed with SeqScape v.2.7 (Applied Biosystems).
Vacuum manifold system
Manufactured by Merck Group
Sourced in United States
The Vacuum Manifold system is a lab equipment product designed to provide a controlled and consistent vacuum environment for various applications. It is a versatile tool that can be used to facilitate various laboratory processes that require a reduced pressure environment.
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2 protocols using vacuum manifold system
1
IFT57 Gene Sequencing Protocol
2
LPMO9J-Catalyzed Substrate Oxidation Assay
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The substrates (0.1%, w/v) were incubated with MtLPMO9J (5 μM) in 20 mM sodium acetate buffer (pH 6.0) at 50°C and shaking at 1000 rpm, in 100 μl total volume for up to 16 h. In all experiments, 1 mM ascorbic acid was used as reducing agent. For the time course experiments, 100 μL aliquots were removed from reactions with 1 mL total volume at defined time intervals. Control reactions were set up without reducing agent or enzyme; reactions containing substrate only were also set up. As a positive control, NcLPMO9C was used [23 (link)]. Reactions were stopped by boiling for 10 min, then the supernatants were separated from insoluble substrates by filtration using a 96-well filter plate (Millipore, MA, USA) and a vacuum manifold system (Millipore). The samples were analyzed by HPAEC-PAD and/or MALDI-TOF/MS (for details, see below).
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