All PCR reactions included a negative control (no DNA) and products were visualized by electrophoresis in 1.5% agarose gel in 1 × TAE buffer containing ethidium bromide (0.5 µg/ml). Prior to sequencing, the PCR products were either gel-extracted using Purelink gel extract kit (Invitrogen) or purified with exonuclease I and shrimp alkaline phosphatase (Thermo Fischer). Purified fragments were directly sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems). All samples were sequenced at least twice from two independent PCRs (ranging from 2 to 5 ×). In order to phase alleles from heterozygote samples, DNA fragments (DRB1 cDNA) were cloned using the TOPO TA cloning kit (Invitrogen) and transformed into competent JM109 cells. Positive clones were confirmed for the presence of correct insert by colony PCR and by enzymatic digestion with EcoR I endonuclease (Thermo Fischer). Plasmid DNA from five clones was purified with EZ-10 Spin Column Plasmid DNA minipreps kit (Bio Basic, Canada) and sequenced using M13 forward and reverse primers.
Exonuclease 1 and shrimp alkaline phosphatase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Exonuclease I and shrimp alkaline phosphatase are enzymes used in molecular biology and genetics research. Exonuclease I removes single-stranded DNA from the 3' end, while shrimp alkaline phosphatase removes 5' phosphate groups from DNA and RNA. These enzymes are often used together to prepare DNA samples for subsequent analysis or manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Abi 3500xl, by Thermo Fisher Scientific (2 mentions)
Abi prism 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ez 10 spin column plasmid dna minipreps kit, by Bio Basic (1 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Hot firepol dna polymerase, by Solis BioDyne (1 mentions)
4 protocols using exonuclease 1 and shrimp alkaline phosphatase
1
Sequencing of Heterozygous DRB1 Alleles
2
Complete Mitochondrial Genome Sequencing
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The complete mtDNA genome was amplified and sequenced by using 21 different sets of primers (Hassanin et al., 2009) . In addition to the available set of primers, 3 fragments did not successfully amplify, therefore, we designed novel primers to cover the gaps (Supplementary file: ST1). DNA amplifications were performed in 20 μl reaction volume containing 1 × PCR buffer (50mM KCl, 10mM Tris-HCl, and pH 8.3), 1.5mM MgCl 2 , 0.2mM of each dNTPs, 3 pmol of each primer, 5U of DreamTaq DNA Polymerase and 1 μl (~30 ng) of template DNA.
The PCR protocol was set as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 40 s and extension at 72 °C for 75 s. The final extension was undertaken at 72 °C for 10 min. PCR amplification was confirmed by electrophoresis and visualized under UV transilluminator. To remove unwanted residual primers, the PCR products were then treated with Exonuclease-I and Shrimp Alkaline Phosphatase (Thermo Scientific Inc.) at 37°C for 20 min and at 85°C for 15 min. The purified fragments were sequenced directly in an Applied Biosystems Genetic Analyzer, ABI 3500 XL in both directions.
The PCR protocol was set as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 40 s and extension at 72 °C for 75 s. The final extension was undertaken at 72 °C for 10 min. PCR amplification was confirmed by electrophoresis and visualized under UV transilluminator. To remove unwanted residual primers, the PCR products were then treated with Exonuclease-I and Shrimp Alkaline Phosphatase (Thermo Scientific Inc.) at 37°C for 20 min and at 85°C for 15 min. The purified fragments were sequenced directly in an Applied Biosystems Genetic Analyzer, ABI 3500 XL in both directions.
3
Sequencing protocol of ANXA5 gene promoter
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Genomic region encompassing the M2 polymorphisms within the minimal promoter region of the ANXA5 gene according to [10 (link)] was amplified from 100 ng of genomic DNA of Estonian subjects using primers PCRI_fw (5’-ACCACGCTCTCCTCTCCAG-3’ ) and PCRI_rev (5’-CCACGCACTATGTTGAGCAC-3’ ; S1 Fig ) and HOT FIREPol DNA Polymerase (Solis Biodyne, Estonia). The obtained PCR product (570 bp) was purified by treating with exonuclease I and shrimp alkaline phosphatase (Thermo Scientific, USA) and subjected to re-sequencing using primer A5_seq (5’-TGGTCGCAGCCCGGGG-3’ ) and BigDye Terminator kit (Applied Biosystems, USA).
The obtained DNA sequences were analyzed using the Phred, Phrap and Consed package [25 ], which enables accurate base calling, sequence quality assessment and assembly. Polymorphisms were identified using the PolyPhred program (ver. 6.02;http://droog.gs.washington.edu/polyphred/ ) and confirmed by manual checking.
The obtained DNA sequences were analyzed using the Phred, Phrap and Consed package [25 ], which enables accurate base calling, sequence quality assessment and assembly. Polymorphisms were identified using the PolyPhred program (ver. 6.02;
4
Complete Mitochondrial Genome Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The complete mtDNA genome was amplified and sequenced by using 21 different sets of primers (Hassanin et al., 2009) . In addition to the available set of primers, 3 fragments did not successfully amplify, therefore, we designed novel primers to cover the gaps (Supplementary file: ST1). DNA amplifications were performed in 20 μl reaction volume containing 1 × PCR buffer (50mM KCl, 10mM Tris-HCl, and pH 8.3), 1.5mM MgCl 2 , 0.2mM of each dNTPs, 3 pmol of each primer, 5U of DreamTaq DNA Polymerase and 1 μl (~30 ng) of template DNA.
The PCR protocol was set as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 40 s and extension at 72 °C for 75 s. The final extension was undertaken at 72 °C for 10 min. PCR amplification was confirmed by electrophoresis and visualized under UV transilluminator. To remove unwanted residual primers, the PCR products were then treated with Exonuclease-I and Shrimp Alkaline Phosphatase (Thermo Scientific Inc.) at 37°C for 20 min and at 85°C for 15 min. The purified fragments were sequenced directly in an Applied Biosystems Genetic Analyzer, ABI 3500 XL in both directions.
The PCR protocol was set as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 40 s and extension at 72 °C for 75 s. The final extension was undertaken at 72 °C for 10 min. PCR amplification was confirmed by electrophoresis and visualized under UV transilluminator. To remove unwanted residual primers, the PCR products were then treated with Exonuclease-I and Shrimp Alkaline Phosphatase (Thermo Scientific Inc.) at 37°C for 20 min and at 85°C for 15 min. The purified fragments were sequenced directly in an Applied Biosystems Genetic Analyzer, ABI 3500 XL in both directions.
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