The assay was performed using as substrate 100 nM of the indicated 2′-deoxyuridine-containing fluorescently labeled probe, that was treated with E. coli UDG (2 U) for 60 min at 37°C in 30 mM HEPES (pH 7.5) and 4% glycerol to obtain natural AP sites. Afterwards, the mixture was supplemented with 5 mM MnCl2 and 10 μM of GTP. The AP lyase activity was started with the addition of 400 nM of the wild-type BsuLigD, in a final volume of 20 μl. During the incubation at 30°C, fluorescence intensity was acquired once per cycle (20 s per cycle) in a CFX384 Real Time System C1000 Thermal Cycler (Bio-Rad), in hard-Shell® 384-Well PCR Plates White Well Clear shell (Bio-Rad CN HSP-3805).
Hard shell 384 well pcr plates white well clear shell
Manufactured by Bio-Rad
The Hard-Shell® 384-Well PCR Plates White Well Clear shell is a laboratory equipment product designed for performing polymerase chain reaction (PCR) experiments. It features 384 well plates with a white well and a clear shell.
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Lab products found in correlation
Cfx384 real time system c1000 thermal cycler, by Bio-Rad (2 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Direct zol rna miniprep plus kit, by Zymo Research (1 mentions)
2 protocols using hard shell 384 well pcr plates white well clear shell
1
Fluorescent Probe-Based AP Lyase Assay
2
Comprehensive RNA Extraction and Gene Expression Analysis
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Total RNA extraction was carried out with Direct-zol RNA MiniPrep Plus kit (ZYMO Research) following manufacturer's instructions. RT reactions were performed using Super Script IV VILO Master Mix (ThermoFisher-Invitrogen). SsoFast™ EvaGreen® Supermix, (Bio-Rad) reagent was used for qPCR expression profiling, carried out in a CFX384 Real Time System C1000 Thermal Cycler (Bio-Rad), in hard-Shell® 384-Well PCR Plates White Well Clear shell (Bio-Rad). ValidPrime® assay was performed to control the presence of genomic DNA background signal during qPCR expression profiling (Figure S1A-C (Supporting Information)). Relative quantification was performed using the 2-ddCq method (Figure S1D (Supporting Information)), using as reference gene HPRT and as calibrator TCP sample.
Statistical analysis was carried out with GraphPad Prism statistical software (n=3 biological replicates). The same cDNA samples used for qPCR profiling were analyzed by endpoint RT-PCR. PCR was carried out using Platinum™ Taq DNA Polymerase (Invitrogen) in a 2720 Thermal Cycler (Applied Biosystems).
Statistical analysis was carried out with GraphPad Prism statistical software (n=3 biological replicates). The same cDNA samples used for qPCR profiling were analyzed by endpoint RT-PCR. PCR was carried out using Platinum™ Taq DNA Polymerase (Invitrogen) in a 2720 Thermal Cycler (Applied Biosystems).
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