Levels of A, C, G and T deoxynucleotide-triphosphates in cells were measured as previously described [5 (link), 71 (link)]. Briefly, U937 cells transduced with SAMHD1-YFP WT and mutants, were sorted for YFP positive cells on a MoFlo™ XDP cell sorter (Beckman) and the cell lysates from 2 × 105 cycling or 4 × 106 differentiated cells were analysed for each dNTP. Levels were quantified by radiolabel incorporation assays, performed using the oligonucleotide templates detailed in [72 (link)] and the procedures described in [73 (link)] with the following modifications: Standard curves ranged from 0.05 to 4 pmol, 1 unit of Taq DNA polymerase (Invitrogen) was used in place of Thermo-Sequenase (GE Healthcare) and 2.5 μM of α-32P-dGTP (to measure dATP, dCTP, and TTP) and α-32P-TTP (to measure dGTP) were employed as an incorporation label. The result was standardized as the pmole amount of each dNTP per 106 cells.
Thermo sequenase
Manufactured by GE Healthcare
Sourced in United Kingdom
Thermo-Sequenase is a thermostable DNA polymerase enzyme used in DNA sequencing applications. It is designed to perform highly accurate and efficient DNA sequencing reactions.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
Kod polymerase, by Merck Group (1 mentions)
Therminator 2, by New England Biolabs (1 mentions)
Oligo clean concentrator, by Zymo Research (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Alconox detergent, by Merck Group (1 mentions)
4 protocols using thermo sequenase
1
Quantification of Cellular Deoxynucleotide Levels
2
Quantification of Cellular dNTP Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular deoxynucleoside triphosphates were extracted from batches of 4x106 differentiated native and SAMHD1 transduced U937 cells according to [40 (link)]. The dNTP levels were quantified by radiolabel incorporation assays performed using oligonucleotide templates detailed in [41 (link)] and the procedures described in [42 (link)] with the following modifications. Standard curves ranged from 0.05 to 4 pmole, 1 unit of KOD polymerase (Merck Millipore) was used in place of Thermo-Sequenase (GE Healthcare) and 2.5 μM of α-32P-dATP was employed as an incorporation label.
3
Synthesis and MALDI-TOF Analysis of Modified Oligonucleotides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides were purchased from Integrated DNA Technologies (IDT Inc.). The 20 µl extension reactions consisted of 3 µM DNA template and 5 µM DNA primer (sequences shown in Fig. 3 ), 10 µM 2’‐F,Me‐UTP (Sierra Bioresearch), 1x Thermo Sequenase buffer or 1x ThermoPol buffer (for Therminator enzymes), and either 10 U Thermo Sequenase (GE Healthcare), 4 U Therminator II or 10 U Therminator IX (New England Biolabs). The 1x Thermo Sequenase buffer consists of 26 mM Tris‐HCl, pH 9.5, and 6.5 mM MgCl2. The 1x ThermoPol buffer contains 20 mM Tris‐HCl, pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, and 0.1% Triton X‐100. Incubations were performed in a thermal cycler using 15 cycles of 30 sec each at 65°C, 45°C, and 65°C. Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI‐TOF‐MS (Bruker ultrafleXtreme) analysis, following a previously described method.29
4
Screening of North African Usher Syndrome Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Screening of known North African mutations associated with USH was performed with a cost-effective NADf chip using multiplex-PCR coupled with dual-color arrayed primer extension as described by Chakchouk et al. [16 (link)]. Multiplex-PCR was performed in a total volume of 50 μl, including 40 μl of the 12 multiplex PCR fragmented products, 0.5 μl of each fluorescent ddNTP, and 1 U of thermosequenase (GE Healthcare, Chalfont St. Giles, UK). Arrays were hybridized with the reaction mixture for 30 min at 60 °C using the FAST Frame Cassette (Sigma-Aldrich, Dorset, UK). The arrays were then washed briefly with 0.3% Alconox detergent (Sigma-Aldrich, Munich, Germany) and distilled water. Slides were then dried before scanning. The coding exons and flanking intronic sequences of all three USH1 genes (MYO7A, USH1C, and PCDH15) were amplified using forward and reverse primers. Mutations were identified by sequencing the PCR products from one affected individual from each family on an ABI Prism 3100-Avant automated DNA Analyzer (Applied Biosystems). Exons harboring mutations were amplified on 40 ng genomic DNA, and then either Sanger sequencing or PCR-restriction fragment length polymorphism (RFLP) analysis was used to examine whether the mutations segregated with the disease in the families and were not present in the 50 control individuals.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!