Nuclear extracts were treated with 10 20 U FastAP (Thermo Scientific) and FastAP buffers (10×) and incubated at 37°C for 1 h. For the control, the nuclear extracts were incubated under the same conditions without FastAP. To verify that PRP18a dephosphorylation is dependent on the concentration of CYP18-1, the density of A. tumefaciens cells harboring CYP18-1 or PPIL3b in was adjusted to 0, 0.1, 0.2, 0.5, and 1.0 at optical density at 600 nm (OD 600 ), and the density of A. tumefaciens cells harboring PRP18a was set to 1.0 at OD 600 using a spectrometer. Infiltration buffer was used to replace the volume of CYP18-1 or PPIL3b as needed so that the final density of PRP18a was the same for each sample. A. tumefaciens cells harboring CYP18-1 or PPIL3b and PRP18a were mixed prior to being introduced into N. benthamiana for simultaneous expression.
Fastap
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, Lithuania
FastAP is a thermosensitive alkaline phosphatase used for the dephosphorylation of DNA and RNA. It catalyzes the removal of 5' phosphate groups from DNA, RNA, and nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (10 mentions)
Exo 1, by Thermo Fisher Scientific (9 mentions)
Exonuclease 1, by Thermo Fisher Scientific (8 mentions)
T4 pnk, by New England Biolabs (8 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Quick ligase, by New England Biolabs (5 mentions)
Nuclease p1, by Merck Group (5 mentions)
Qiaquick gel extraction kit, by Qiagen (4 mentions)
T4 pnk, by Thermo Fisher Scientific (4 mentions)
γ 32p atp, by PerkinElmer (4 mentions)
5 deadenylase, by New England Biolabs (3 mentions)
T4 polynucleotide kinase, by New England Biolabs (3 mentions)
Q5 dna polymerase, by New England Biolabs (3 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Rnase 1, by Thermo Fisher Scientific (3 mentions)
T4 rna ligase, by New England Biolabs (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (3 mentions)
Abi 3730xl, by Thermo Fisher Scientific (3 mentions)
Primer 5, by Premier Biosoft (3 mentions)
123 protocols using fastap
1
PRP18a Dephosphorylation by CYP18-1
2
Quantifying RNA Nucleoside Modifications
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For each sample, 25 to 50 ng of RNA was digested with 1 μl Nuclease P1 (Sigma) in P1 buffer (25 mM NaCl, 2.5 mM ZnCl2) in a final reaction volume of 20 μl for 2 h at 42 °C. Subsequently, 1 μl of FastAP (Thermo Fisher) and 2.5 μl of 10x FastAP buffer were added to each sample, and they were incubated at 37 °C for 4 h. Samples were then diluted with an equal volume of water and filtered through a 0.2 μm PVDF filter (0.2 μm pore size, 0.4 mm diameter, Millipore). Five microliters of each filtered sample was separated by reverse-phase ultraperformance liquid chromatography on a ZORBAX Eclipse XDB-C18 Rapid Resolution HT 2.1 × 50 mm, 1.8 μm column (Agilent) on an Agilent Technologies 1290 Infinity II liquid chromatography system, followed by mass spectrometry on a Sciex Triple Quad 6500 triple-quadrupole mass spectrometer in positive electrospray ionization mode. Nucleosides were quantified using nucleoside-to-base transitions of 282.101 > 150.100 (m6A), 282.101 > 150.100 (m1A), 284.982 > 153.100 (d3m6A), 284.983 > 153.200 (d3m1A), 296.101 > 164.100 (m6,6A), 267.966 > 136.000 (A), and 284.004 > 152.100 (G). Standard curves were generated by injecting known concentrations of the corresponding pure nucleosides in the same run, and the percentage of modified to unmodified nucleosides was calculated based on the calibrated concentrations.
3
Radioactive cOA Degradation Analysis
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The cOA degradation reaction, containing 1 mL of radioactive cOA, 20 mM Mes (pH 6.0), 5 mM MnCl 2 , 5 mM DTT and 2 mL of 7#, was stopped at desired time points by cooling on ice. Then, aliquots of the reaction mixtures were added into 10 mL FastAP reaction mixtures, containing 1 mL of 10X Buffer and 1 mL FastAP (Thermo Fisher Scientific, Waltham, MA, USA), which were then incubated at 37 C for 30 min and analyzed by denaturing gel electrophoresis. Mock treatment using water instead of FastAP was performed as controls. In addition, the cOA cleavage products by SiRe_0244 were also treated with FastAP by a similar procedure.
4
Dual Protein Expression Plasmid Construction
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First, the HsSPATA22-1-363 coding sequence was amplified from pET15b-HsSPATA22-1-363 (obtained by subcloning HsSPATA22-1-363 from pCMV6-XL5- HsSPATA22 (Origene, cat# SC123038 with Nde1 and BamHI) with SP0636 and SP0637 primers. The resulting PCR product was digested by MscI + BglII and ligated in the SC0583 plasmid digested by the same enzymes and treated with Fast-AP (Alkaline Phosphatase, ThermoFisher), thus generating pCIG-MCS-IRES-HsSPATA22-1-363 plasmid. Then, the HsMEIOB-1-471-cMyc-Flag coding sequence was amplified from pCMV6-HsMEIOB-1-471 (Origene, cat# RC228391) with SP0633 and SP0635. The resulting PCR product was digested by EcoRI-HF + XmaI and ligated in pCIG-MCS-IRES-HsSPATA22-1-363 digested by the same enzymes and treated with Fast-AP (ThermoFisher) generating pCIG-HsMEIOB-1-471-cMyc-Flag-IRES-HsSPATA22-1-363 (SC0585 plasmid).
5
DNA Extraction and SNP Genotyping
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Peripheral blood samples were collected, and DNA was extracted from leukocytes by using phenol–chloroform isopropyl alcohol method. Primers were designed by Primer 5 (version 5.00, PREMIER Biosoft International). After purification of polymerase chain reaction (PCR) products by both phosphorylase (FastAP, Applied biosystems) and exonuclease I (EXO I, Applied biosystems), extension reaction was done by SNaPshot Multiplex kit of ABI. The extended products were further purified by phosphorylase (FastAP, Applied biosystems), and sampled by ABI3730xl (Applied biosystems). The results of SNP typing were analyzed by genemap 4.0 (Applied biosystems).
The primer information is as follows:
Forward: TCC TGG GGT GTT TGA ATC ATA AG
Reverse: TGT GTT CAC ACA AAA TGT GGG ATT A
Elongation: TGC TTT TTA GAA AAA TAT CAG GAA C.
The primer information is as follows:
Forward: TCC TGG GGT GTT TGA ATC ATA AG
Reverse: TGT GTT CAC ACA AAA TGT GGG ATT A
Elongation: TGC TTT TTA GAA AAA TAT CAG GAA C.
6
SNP Genotyping of Alzheimer's Biomarkers
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The genomic DNA was extracted from blood (2 ml) stored in an ethylene diamine tetraacetie acid (EDTA) anticoagulation tube using the phenol-chloroform-isopropyl alcohol method. Polymerase chain reaction (PCR) and extension primers scheme were achieved through Primer 5 software (PREMIER Biosoft International, Version 5.00). PCR materials were conducted by purification with phosphorylase (FastAP, Applied Biosystems) and exonuclease I (EXO I, Applied Biosystems). A consequent extension was applied by the ABI SNaPshot Multiplex Kit (Applied Biosystems). Extended products were purified with FastAP and loaded into ABI3730xl (Applied Biosystems). GeneMapper 4.0 (Applied Biosystems) was used to conduct data analysis. The SNaPshot technique (Applied Biosystems) was utilized for genotyping of SNPs. The following SNPs were tested: rs10792832, rs11136000, rs11218343, rs1990620, rs1990622, rs3173615, rs34860942, rs3764650, rs3792646, rs3818361, rs3851179, rs4147929, rs56081887, rs5848, rs6656401, rs6701713, rs6733839, rs704180, rs744373, rs9331888, and rs9637454. The SNPs rs429358 and rs7412 of the APOE gene were determined by Sanger sequencing. Details of primers were described in Supplementary Table 1 .
7
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 0.5% Tween-20, 1% Nonidet P-40, and protease inhibitors for 20 min on ice. Lysates were cleared by centrifugation, and soluble protein was used for immunoprecipitation or mixed with 2 Laemmli Sample Buffer (Bio-Rad) and incubated for 7 min at 96 °C and analyzed by Western blot. For immunoprecipitation, protein extracts were incubated with GFP-Trap beads (ChromoTek) at 4 °C for 120 min. Beads were washed five times with lysis buffer and incubated with 2 Laemmli Sample Buffer (Bio-Rad) for 7 min at 96 °C. In case of phosphatase treatment, washed beads after IP were incubated with 10 U of FastAP (Fermentas) in 1 FastAP buffer at 37 °C for 30 min, pelleted, and incubated with Laemmli Sample Buffer. Proteins were resolved in 4–12% Bis-Tris or 3–8% Tris–acetate gels (Life Technologies), transferred to 0.45- m nitrocellulose membrane (Bio-Rad), and immunoblotted.
8
5′ Phosphorylation of RNA Transcripts
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In vitro transcribed RNA (30 pmol) was dephosphorylated by 0.1 U/µL FASTAP (Fermentas) in 1× PNK A buffer (Fermentas) for 30 min at 37°C before FASTAP was heat-inactivated for 20 min at 75°C. Subsequently, 30 µCi γ32P-ATP (Hartmann Analytic) and T4 PNK (final concentration 0.5 U/µL; Fermentas) were added, and the 5′ phosphorylation reaction was carried out in 1× PNK A buffer for 30 min at 37°C. T4-PNK was heat-inactivated for 10 min at 75°C. Free γ32P-ATP was removed by desalting on a G25 spin column (GE Healthcare) according to the manufacturer's instructions. 5′-32P-labeled RNA was stored at −20°C until further use. Chemically synthesized small RNAs (30 pmol; Biomers) were directly phosphorylated by T4-PNK following the same protocol.
9
Physcomitrium patens PPR Protein Editing
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Plasmids based on pETG_41K_MCS harboring the coding sequences of wild-type Physcomitrium patens editing factors PPR56 and PPR65 and their 46 bp targets (28 (link)) were used to amplify PPR protein target combinations using Q5 polymerase (New England Biolabs) and primers with restriction site overhangs (Supplementary Table S1 , Integrated DNA Technologies). After digest (FastDigest enzymes ApaI and ScaI, Thermo Fisher Scientific; CutSmart enzymes MscI and NotI, New England Biolabs), the constructs were introduced into the dephosphorylated (FastAP, Thermo Fisher Scientific) eukaryotic expression vectors pEYFP-C1 and pCMV-HA (Clontech TaKaRa), respectively, to create the final fusion protein coding sequences. Plasmid DNA for all constructs was isolated and purified using the NucleoBond® Xtra Midi kit (Macherey Nagel). Construct sequences were verified by Sanger Sequencing (Macrogen Europe). To generate constructs with coding sequence mutations, rolling-circle (38 ) or overlap extension PCRs (39 (link)) were performed (for oligonucleotides see Supplementary Table S1 ). E. coli RNA editing experiments with mutated PPR protein versions inserted in petG_41K_MCS with their 46 bp targets were performed as outlined in (28 (link)).
10
Verification of Real-time PCR Amplicon Identity
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Real-time PCR products were subsequently sequenced to verify the identity of the amplified sequences. Sanger sequencing was performed on the same amplicons as used for HRM analysis. 5 μl of PCR products were purified with exonuclease I and Fast-AP (Thermo Fisher Scientific, Waltham, USA) for 15 min at 37 °C and 15 min by 80 °C. A sequencing reaction was set up with 1 μl of purified PCR products and the BigDye® Terminator v1.1 Cycle Sequencing Kit (Life Technologies). Briefly, each 20 μl sequencing reaction mixture contained 1 μl of PCR amplicon, 0.16 μM of either forward or reverse PCR primer, 0.5 μl of BigDye Ready Reaction Mix, and 1 X sequencing buffer. The sequencing reaction conditions were as follows: 30 cycles of denaturing at 96 °C for 10 seconds, annealing at 50 °C for 5 seconds, and extension at 60 °C for 4 minutes. The sequencing products were purified using ethanol/EDTA/sodium acetate precipitation. Sequencing was performed on an ABI 3130xl 16-capillary automated genetic analyser.
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