Exo resistant random primer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Exo-Resistant Random Primer is a laboratory tool designed for use in various molecular biology applications. It is a short, synthetic DNA sequence that can be used to initiate DNA synthesis in a non-specific manner. The primer is engineered to be resistant to exonuclease activity, ensuring stability and reliability during experimental procedures.

Automatically generated - may contain errors

Lab products found in correlation

13 protocols using exo resistant random primer

Whole Genome Amplification by MDA

Check if the same lab product or an alternative is used in the 5 most similar protocols

DNA was denatured by mixing the DNA diluted in milliQ water 1:1 with 50 mM KOH (Sigma Aldrich) and incubating for 3 min at room temperature (RT). The denatured DNA was the neutralized by adding an equal volume of Tris-HCl (80 mM, pH4; Sigma Aldrich). RepliPHI Phi29 Reagent Kit (Epicenter) supplemented with Exo-Resistant Random Primer (ThermoFisher Scientific) was used for the MDA reaction. A 2× MDA mastermix (2× reaction buffer, 2 mM dNTP, 50 μM primer, 4 U/μl Phi29, 8 mM DTT and 5 % DMSO) was prepared. The denatured and neutralized DNA and the 2× MDA mastermix were mixed at equal volumes by pipetting for a bulk reaction in tube or in the microfluidic chip as described above for emulsion generation. Reactions were incubated for 12 h at 30 °C. The polymerase was then inactivated at 65 °C for 10 min.
After incubation, the emulsion was broken by adding 5 μl 1H, 1H, 2H, 2H, Perfluoro-1-octanol (Sigma Aldrich), vortexing, and centrifuging briefly until the emulsion separated into one aqueous and one oil phase. If the emulsion did not break, the emulsion breaking procedure was repeated. The supernatant (aqueous phase) was collected by pipetting and could then be treated like the MDA products from the bulk reactions. The concentrations of MDA products were quantified with Qubit dsDNA kit (ThermoFisher Scientific) or Quant-iT PicoGreen dsDNA assay (ThermoFisher Scientific).

Hammond M., Homa F., Andersson-Svahn H., Ettema T.J, & Joensson H.N. (2016). Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis. Microbiome, 4, 52.

+ Open protocol

+ Expand

Single-Cell Whole-Genome Amplification Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

We amplified multiple single B lymphocytes from each of 14 humans according to our SCMDA protocol (5 (link)). In brief, 1 µL of Exo-Resistant random primer (Thermo Fisher Scientific) was added to the cell, followed immediately by 3 µL of lysis buffer (400 mM KOH, 100 mM DTT, and 10 mM EDTA). Cell lysis and DNA denaturation were performed on ice for 10 min. Then 3 µL of stop buffer (400 mM HCl and 600 mM Tris⋅HCl pH 7.5) was added to neutralize the lysis buffer. Finally, 32 µL of Master Mix containing 30 µL of MDA reaction buffer and 2 µL of Phi29 polymerase (REPLI-g UltraFast Mini Kit; Qiagen) were added. SCMDA was carried out for 1.5 h at 30 °C, 3 min at 65 °C, and holding at 4 °C until purification. SCMDA product was purified using AMPureXP-beads (Beckman Coulter), and the concentration was measured with the Qubit High-Sensitivity dsDNA Kit (Thermo Fisher Scientific). Meanwhile, 1 ng of human genomic DNA in 2.5 µL of PBS was amplified as a positive control, and 2.5 µL of PBS without any template was amplified as a negative control. All single-cell amplicons of sufficient yield were subjected to a locus dropout test (5 (link)), after which a total of four amplicons per donor that passed the test were prepared for whole-genome sequencing.

Zhang L., Dong X., Lee M., Maslov A.Y., Wang T, & Vijg J. (2019). Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan. Proceedings of the National Academy of Sciences of the United States of America, 116(18), 9014-9019.

+ Open protocol

+ Expand

Single-Cell Multiple Displacement Amplification

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

We amplified the isolated single cells using the Single-Cell Multiple Displacement Amplification protocol reported previously (6 (link)). Briefly, for each single cell, we added 1 μl of exo-resistant random primer (Thermo Fisher Scientific) and 3 μl of lysis buffer (400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA) and incubated on ice for 10 min. We neutralized the lysis buffer by adding 3 μl of stop buffer [400 mM HCl and 600 mM tris-HCl (pH 7.5)]. We then added 32 μl of master mix containing 30 μl of multiple displacement amplification reaction buffer and 2 μl of Phi29 polymerase (REPLI-g UltraFast Mini Kit, QIAGEN), incubated for 1.5 hours at 30°C and 3 min at 65°C, and held at 4°C until purification. We purified the amplicons using AMPure XP beads (Beckman Coulter) and quantified DNA concentration with the Qubit High-Sensitivity dsDNA Kit (Thermo Fisher Scientific). Simultaneously, we amplified 1 ng of genomic DNA in 2.5 μl of PBS as positive control and 2.5 μl of PBS without any template as negative control. We performed the locus dropout test as described previously (6 (link)) with primers designed for each species separately (table S2). Three single-cell amplicons per individual per experimental condition that passed the locus dropout test were prepared for whole-genome sequencing.

Zhang L., Dong X., Tian X., Lee M., Ablaeva J., Firsanov D., Lee S.G., Maslov A.Y., Gladyshev V.N., Seluanov A., Gorbunova V, & Vijg J. (2021). Maintenance of genome sequence integrity in long- and short-lived rodent species. Science Advances, 7(44), eabj3284.

+ Open protocol

+ Expand

Random Primer Labeling of DNA Probes

Check if the same lab product or an alternative is used in the 5 most similar protocols

The DNA probe obtained by microdissection procedures was labelled in a DOP-PCR reaction in the presence of TAMRA-5-dUTP as described earlier [23 (link)]. Fragments obtained by gene-specific PCR were labelled using a Random Primer Labelling protocol: 25 μl of labelling reaction contained 50 ng DNA, 1× Klenow buffer (Thermo Fisher Scientific), 44 ng/μl Exo-Resistant Random Primer (Thermo Fisher Scientific) 0.1 mM dATP, dGTP, dCTP, and 0.015 mM dTTP, 0.016 mM TAMRA-5-dUTP, or Biotin-11-dUTP (Biosan, Novosibirsk, Russia), and 5 U of Klenow fragment (Thermo Fisher Scientific TM) in a PCR tube. The required amounts of DNA, Klenow buffer, and Random Primers were mixed, brought up to 12 μl with water, and heated at 95 °C for 5 min in a thermocycler. The solution was chilled on ice, and appropriate amounts of nucleotides, Klenow fragment, and water were added to reach 25 μl. The reaction mix was incubated at 37 °C for 18 h. Fluorescence in situ hybridization (FISH) was performed using a previously described standard protocol [25 , 27 ].

Artemov G.N., Gordeev M.I., Kokhanenko A.A., Moskaev A.V., Velichevskaya A.I., Stegniy V.N., Sharakhov I.V, & Sharakhova M.V. (2018). A standard photomap of ovarian nurse cell chromosomes and inversion polymorphism in Anopheles beklemishevi. Parasites & Vectors, 11, 211.

+ Open protocol

+ Expand

Random Primer Labeling DNA-Probes for FISH

Check if the same lab product or an alternative is used in the 5 most similar protocols

Gene-specific DNA-probes were labelled using a Random Primer Labeling Protocol: 25 μl of labelling reaction contained 50-ng DNA, 1x Klenow buffer (Thermo Fisher Scientific TM, USA), 44-ng/μl Exo-Resistant Random Primer (Thermo Fisher Scientific TM, USA) 0.1-mM dATP, dGTP, and dCTP and 0.015-mM dTTP, 0.016-mM TAMRA-5-dUTP, and 5 units of Klenow fragment (Thermo Fisher Scientific TM, USA) in a PCR tube. The required amounts of DNA, Klenow buffer, and Random Primers were mixed, brought up to 12 μl with water, and heated at 95 °C for 5 min in a thermocycler. The solution was chilled on ice, and appropriate amounts of nucleotides, Klenow fragment and water were added to reach 25 μl. The reaction mix was incubated at 37 °C for 18 h. FISH was performed using previously described standard protocol [39 (link), 40 ].

Artemov G.N., Velichevskaya A.I., Bondarenko S.M., Karagyan G.H., Aghayan S.A., Arakelyan M.S., Stegniy V.N., Sharakhov I.V, & Sharakhova M.V. (2018). A standard photomap of the ovarian nurse cell chromosomes for the dominant malaria vector in Europe and Middle East Anopheles sacharovi. Malaria Journal, 17, 276.

+ Open protocol

+ Expand

Optimization of TN-RCA RNA Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

The TN-RCA reaction product was digested with MseI (NEB) (10 U) for 20 min at 37 °C in the presence or absence of cutting primer, Msecutprimer: 5’-TTTATCTTAACTCACCAACT-3’ (underlined: MseI recognition site), and the enzyme subsequently inactivated at 65 °C for 20 min. Lambda exonuclease (NEB) (0.5 U), exonuclease III (NEB) (20 U), exonuclease VIII (NEB) (2 U), T7 exonuclease (2 U), RNase H (NEB) (1 U), RNase A (Thermo Scientific, Waltham, MA, USA) (10 U), RNase A/T1 Mix (Thermo Scientific) (1 U), RNase T1 (Invitrogen/Ambion) (1 U), ShortCut RNase III (NEB) (0.4 U) were added to the TN-RCA reaction mixture after the ligation step was completed, or at various times during the TN-RCA reaction. In some experiments, ShortCut RNase III (NEB) (0.4 U) was also added before the ligation step to fragment the target genomic Zika RNA. Random hexamers (Exo-Resistant Random Primer; 0.2 μL of 500 μM stock; Thermo Scientific) also was added after the ligation step.

Zingg J.M, & Daunert S. (2018). Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA. Methods and Protocols, 1(2), 15.

+ Open protocol

+ Expand

Whole Genome Amplification via phi29

Check if the same lab product or an alternative is used in the 5 most similar protocols

40ng of human genomic DNA was mixed with 2 µL of 10X phi29 reaction buffer and 2 µL of exo-resistant random primer (Thermo Scientific) in a 17 µL reaction. The mixture was incubated at 95°C for 5min and then gradually cooled to 30°C (1°C/15 s). 0.5 µL dNTP mix (10mM), 0.5 µL BSA (10mg/ml), 1 µL phi29, and 1 µL inorganic pyrophosphatase (Thermo Scientific) were added to the reaction. The mixture was then incubated for 6 h at 30°C. High molecular weight amplification products were observed on an agarose gel, and the amplicons were purified using standard ethanol precipitation.

Pal M., Ebrahimi S., Oh G., Khare T., Zhang A., Kaminsky Z.A., Wang S.C, & Petronis A. (2015). High Precision DNA Modification Analysis of HCG9 in Major Psychosis. Schizophrenia Bulletin, 42(1), 170-177.

+ Open protocol

+ Expand

Random Rolling Circle Amplification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Random rolling circle amplification was performed as previously described (7 (link)) with some modifications. A 20 μl reaction was set up using 5 μl of size-selected template DNA, 1 mM dNTPs, 10 U Phi29 DNA polymerase (EP0092, Thermo Fisher Scientific), 50 μM Exo-resistant random primer (SO181, Thermo Fisher Scientific), 0.02 U inorganic pyrophosphatase (EF0221, Thermo Fisher Scientific) and 1× Phi29 DNA polymerase buffer (supplied with enzyme). The reaction was run at 30°C for 18 h and stopped by heating to 65°C for 2 min. Product DNA was purified by sodium acetate/ethanol precipitation. We also used the illustra TempliPhi 100 amplification kit (25640010, GE Life Sciences) and obtained similar amplification results.

Mehta D., Hirsch-Hoffmann M., Were M., Patrignani A., Zaidi S.S., Were H., Gruissem W, & Vanderschuren H. (2018). A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field. Nucleic Acids Research, 47(2), e9.

+ Open protocol

+ Expand

Single-Cell Genome Amplification via WGA-X

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Prior to single-cell genome amplification, cells were lysed by one freeze-thaw cycle at −80°C and then at 20°C. The amplification procedures followed the WGA-X protocol (53 (link)), except that the total volume was 5 µL instead of 10 µL. In brief, the WGA-X components are as follows: 0.2 U µL⁻¹ Equiphi29 polymerase (Thermo Fisher Scientific), 1

\times

Equiphi29 reaction buffer (Thermo Fisher Scientific), 10 mM dithiothreitol (Thermo Fisher Scientific), 40 mM Exo-Resistant Random Primer (Thermo Fisher Scientific), 0.4 mM dNTP (New England BioLabs), and 1 µM SYTO-13 (Thermo Fisher Scientific). The WGA-X reaction was carried out by using a CFX384 TouchTM Real-Time Detection System (Bio-Rad Laboratories) for 7 hours at 45°C and then inactivated by incubation at 75°C for 15 min.

Lo H.Y., Wink K., Nitz H., Kästner M., Belder D., Müller J.A, & Kaster A.K. (2023). scMAR-Seq: a novel workflow for targeted single-cell genomics of microorganisms using radioactive labeling. mSystems, 8(6), e00998-23.

+ Open protocol

+ Expand

BAC Clone Labelling for Gene FISH

Check if the same lab product or an alternative is used in the 5 most similar protocols

Rolling cycle amplification method was used to label Bacterial Artificial Chromosome (BAC) clone containing zyxin sequence (RP23–358011, from SourceBioScience, UK) with biotin or Dig labelled UTP. For the rolling circle amplification, 100 ng of the BAC clone was denatured at 95°C for 5 min in 10 μl of annealing buffer (80 mM TRIS-HCl, pH 8.0 and 20 mM MgCl₂) before moving to ice. The denatured DNA was subjected to amplification by six units of phi 29 DNA polymerase (New England Biolabs, catalogue no. M0269S) with 0.2 mM d(AGC)TP, 0.15 mM dTTP, and 0.1 mM labelled UTP and 12.5 μM Exo-Resistant Random Primer (Thermo scientific, catalogue no. S0181). The amplification was carried out at 30°C for 8 h, 65°C for 10 min and kept at 4°C till CviKI-1 digestion to generate DNA fragments of 100–400 bp in size. The digested DNA fragments was precipitated using 3M Sodium Acetate and twice the volume of Ethanol for 1 h in −80°C followed by centrifugation at 13 000 rpm for 20 min on a desktop centrifuge. The pellet of the gene FISH probe was washed with 70% ethanol and re-suspended in 10 mM Tris-HCL pH8.0 and used for FISH.

Maharana S., Iyer K.V., Jain N., Nagarajan M., Wang Y, & Shivashankar G.V. (2016). Chromosome intermingling—the physical basis of chromosome organization in differentiated cells. Nucleic Acids Research, 44(11), 5148-5160.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!