The microsatellite DNA regions of interest were selectively recovered using a Magenesphere magnetic separation kit (Promega) and biotin-labeled probes (GT)10 and (CT)10. After the addition of 50 μL of hybridization solution (12× SSC, 0.1% SDS) together with the probes to the linker-ligated DNA, the mixture (100 μL) was heated to 95 °C for 15 min and then incubated at 60 °C for 12 h. Hybridization was performed using Streptavidin paramagnetic particles (Promega) in accordance with the manufacturer’s instructions. Single-stranded DNA was eluted at 95 °C for 15 min in 100 μL of low-TE buffer containing 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA. PCR was performed in mixtures consisting of 10 μL of DNA, 4 μL of 10 μM SNX primer, 0.3 μL of Vent (-exo) polymerase (New England Biolabs), 4 μL of dNTP mix, 5 μL of 10× Thermopol buffer, and 26.7 μL of sterilized distilled water with denaturation at 96 °C for 5 min followed by 40 cycles of 96 °C for 45 s, 62 °C for 1 min, 72 °C for 2 min, and a final elongation step at 72 °C for 5 min.
Vent exo polymerase
Manufactured by New England Biolabs
Vent (exo-) polymerase is a thermostable DNA polymerase derived from the hyperthermophilic archaeon Thermococcus litoralis. It possesses 5' to 3' polymerase activity and lacks 3' to 5' exonuclease proofreading activity.
Automatically generated - may contain errors
4 protocols using vent exo polymerase
1
Microsatellite DNA Enrichment and Amplification
2
G-quadruplex induced replication blocks
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Presence of replication blocks due to G-quadruplex structure formation was studied in the plasmids, pMN7 and pMN8, by primer extension. The reactions were carried out by mixing 100 ng (except when different concentrations of template DNA were used) of DNA sample in 1X Thermo polymerase buffer (10 mM KCl, 10 mM (NH4)2SO4), 20 mM Tris-HCl (pH 8.8), 4 mM MgSO4 and 0.1% Triton X-100), 4 mM MgSO4, 200 μM dNTPs, 0.1 nM end labeled oligomers and 1 U Vent (exo-) polymerase (New England Biolabs). Linear amplification primer extensions were carried out in a PCR machine (20 cycles) under the following conditions: 95°C for 3 min (1 cycle), 94°C for 45 sec, 56°C for 45 sec and 72°C for 45 sec (20 cycles) and final extension of 3 min at 72°C.
3
Phage DNA Nicking and Extension
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First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 µM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation.
4
DNA Template Construction for RNA Transcription
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The DNA constructs to be used as templates for RNA transcription were synthesized by annealing and amplifying the target oligos and the guide oligos (Figure 1 , Supplementary Table S1 ). The annealing/amplification PCR [0.5 μM target oligo, 1.5 μM guide oligo, 1 mM dNTPs in 20 μl total volume] was set up using Vent (exo-) polymerase [0.04 U/μl] (NEB). Because this polymerase lacks 3’ to 5’ and 5’ to 3’ exonuclease activity, the mismatches caused by the randomness in the guide will not be corrected by the polymerase. PCR annealing was set at 55°C for 30 s and run for 30 cycles. Afterwards, PCR products were run on a 2% agarose gel and purified.
The purified products were used as templates for another round of PCR amplification to add the sequence for the P2 primer and obtain the amount needed for RNA transcription. A 2-step PCR reaction [0.8 ng template, 375 nM forward primer, 375 nM reverse primer, 50 μM dNTPs in 40 μl total volume] was set up using the forward synthesis and P2 primers (Supplementary Table S1 ) and Vent(exo-) polymerase [0.02 U/μl]. The PCR products were column purified and the correct assembly was confirmed by agarose gel electrophoresis and Sanger sequencing (Azenta Life Sciences).
The purified products were used as templates for another round of PCR amplification to add the sequence for the P2 primer and obtain the amount needed for RNA transcription. A 2-step PCR reaction [0.8 ng template, 375 nM forward primer, 375 nM reverse primer, 50 μM dNTPs in 40 μl total volume] was set up using the forward synthesis and P2 primers (
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