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Pcr2.1 topo ta vector

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The PCR2.1-TOPO TA vector is a laboratory tool used for the direct cloning of Taq polymerase-amplified PCR products. It provides a simple and efficient method for the insertion of PCR fragments into a plasmid vector.

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Lab products found in correlation

Ecori, by New England Biolabs (3 mentions) Qiaprep spin miniprep kit, by Qiagen (3 mentions) T4 rna ligase, by New England Biolabs (2 mentions) Riboblock rnase inhibitor, by Thermo Fisher Scientific (2 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Random hexamers, by Eurofins (2 mentions) Random hexamer, by Thermo Fisher Scientific (2 mentions) Genelute plasmid miniprep kit, by Merck Group (2 mentions) Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions) T4 pnk enzyme, by New England Biolabs (1 mentions) Nucleobond xtra midi kit, by Macherey-Nagel (1 mentions) M mlv buffer, by Thermo Fisher Scientific (1 mentions) Rna clean concentrator kit, by Zymo Research (1 mentions) Bigdye terminator sequencing kit, by Thermo Fisher Scientific (1 mentions) Dh5α competent cells, by Toyobo (1 mentions) Mirneasy kit, by Qiagen (1 mentions) Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions) Bigdye terminator v3.1 sequencing reagents, by Thermo Fisher Scientific (1 mentions) 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions) Xhoi, by Roche (1 mentions)

Spelling variants of this product

PCR2.1-TOPO vector (271 citations) PCR2.1-TOPO (217 citations) PCR2.1 vector (124 citations) TOPO-TA vector (100 citations) TOPO TA (71 citations) PCR2.1-TOPO-TA (13 citations) PCR2.1 TA vector (10 citations) PCR2-TOPO vector (7 citations) Topo-TA vector pCR2.1 (2 citations) PCR2.1 TA (2 citations)

40 protocols using pcr2.1 topo ta vector

1

Generating Fab' Fragments with CRISPR-Cas9

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The genomic sequence
of the Ighg1 constant heavy chain and Igkc constant light chain were
identified using the Ensembl genome browser (release 98,46 (link) accession numbers – Ighg1: ENSMUSG00000076614,
Igkc: ENSMUSG00000076609). gRNA-H.m1 (5′-CTTGGTGCTGCTGGCCGGGT-3′)
and gRNA-L.mκ (5′-GGAATGAGTGTTAGAGACAA-3′)
were designed using http://crispor.tefor.net/(47 (link)) and ordered as single-stranded oligos
from Integrated DNA Technologies (IDT) with the appropriate BbsI overhangs
for cloning into the plasmid px330-U6-Chimeric_BB-CBh-hSpCas9, which
was obtained as a gift from Feng Zhang (Addgene plasmid #42230). Oligos
were phosphorylated using a thermocycler with the T4 PNK enzyme (M0201,
New England Biolabs) by incubation at 37 °C for 30 min, and annealed
by incubation at 95 °C for 5 min, and cooling at a rate of 0.1
°C/s to 25 °C. Annealed oligos of gRNA-H.m1 and gRNA-L.mκ
were cloned into the px330 vector at the BbsI site. Synthetic gene
fragments containing homologous arms and desired insert for Fab′
fragment generation and tag insertion were synthesized by IDT and
cloned into the PCR2.1 TOPO TA vector (K-450001, ThermoFisher Scientific).
All gRNA/Cas9 and HDR plasmids were isolated from DH5α competent E. coli and purified with the NucleoBond Xtra Midi
Kit (740410.100, Macherey-Nagel) according to the manufacturer’s
protocol.
Le Gall C.M., van der Schoot J.M., Ramos-Tomillero I., Khalily M.P., van Dalen F.J., Wijfjes Z., Smeding L., van Dalen D., Cammarata A., Bonger K.M., Figdor C.G., Scheeren F.A, & Verdoes M. (2021). Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering. Bioconjugate Chemistry, 32(2), 301-310.
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2

PCR Validation of Deletion Junctions

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Once possible deletion junctions were identified bioinformatically they were confirmed using conventional PCR. Primers were designed that flank the junction and did not contain repeats as identified by using Repeat Masker (Smit et al. 2013). The forward primer used was 5’-CAGTTATCTGACTGTAACACTGTAGGC-3’ and the reverse primer used was 5’-GTTGTTGCTTATGCTGGTCTTG-3’ to generate a 650 bp product. For 3 individuals the PCR product was subcloned into pCR 2.1-TOPO TA vector (Thermo Fisher) and Sanger sequenced for confirmation.
Ruhno C., McGovern V.L., Avenarius M.R., Snyder P.J., Prior T.W., Nery F.C., Muhtaseb A., Roggenbuck J.S., Kissel J.T., Sansone V.A., Siranosian J.J., Johnstone A.J., Nwe P.H., Zhang R.Z., Swoboda K.J, & Burghes A.H. (2019). Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype. Human genetics, 138(3), 241-256.
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3

Circular RNA Mapping and Sequencing

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Five microgram of total RNA were circularized with 40 U of T4 RNA ligase (New England Biolabs), following the manufacturer's instructions. Circularized RNA were phenol extracted and precipitated in one volume of isopropanol, for 2h at −20°C. Circularized RNA were resuspended in 14.5 μl of water and first strand complementary DNA (cDNA) synthesis was done for 3 h at 40°C using 400 U of M-MLV reverse transcriptase (Fermentas), 8 mM of random hexamers (Eurofins), 1X M-MLV buffer, 0.5 mM dNTPs and 40 U of Riboblock RNase inhibitor (Fermentas). The obtained cDNA were diluted four times, and 5 μl of the obtained cDNA solution were used for PCR amplification with divergent primers. The primers used for mapping nad1 exon 1, exons 2–3 and exons 4–5 precursor transcripts are listed in Supplementary Table S1. Amplified PCR products were gel purified, cloned into pCR2.1®-TOPO® TA vector (ThermoFisher Scientific) and inserts of independent recombinant plasmids were sequenced after Escherichia coli transformation.
Wang C., Aubé F., Planchard N., Quadrado M., Dargel-Graffin C., Nogué F, & Mireau H. (2017). The pentatricopeptide repeat protein MTSF2 stabilizes a nad1 precursor transcript and defines the 3΄ end of its 5΄-half intron. Nucleic Acids Research, 45(10), 6119-6134.
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4

Sequencing of Circularized RNA Transcripts

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Five μg of total RNA were circularized with 40 U of T4 RNA ligase (New England Biolabs), following the manufacturer's instructions. Circularized RNA was purified using the RNA Clean & Concentrator Kits (Zymo Research®, California, USA). The first strand complementary DNA (cDNA) synthesis was done for 3 h at 40°C using 400 U of M-MLV reverse transcriptase (Fermentas), 8 mM of random hexamers (Eurofins), 1× M-MLV buffer, 0.5 mM dNTPs and 40 U of Riboblock RNase inhibitor (Fermentas). The obtained cDNA was diluted four times, and 5 μl of the obtained cDNA solution was used for PCR amplification with divergent primers. The primers used for mapping precursor transcripts are listed in Supplementary Table S1. Amplified PCR products were gel purified, cloned into pCR2.1®-TOPO® TA vector (ThermoFisher Scientific) and inserts of independent recombinant plasmids were sequenced after E. coli transformation.
Wang C., Quadrado M, & Mireau H. (2023). Interplay of endonucleolytic and exonucleolytic processing in the 3′-end formation of a mitochondrial nad2 RNA precursor in Arabidopsis. Nucleic Acids Research, 51(14), 7619-7630.
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5

Molecular Analysis of Barley Endosperms and Leaves

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Plant materials for molecular analysis were collected from barley (H. vulgare cultivar Golden Promise) endosperms of T0 and leaves of T0 and T1 progeny. Genomic DNA was isolated using the CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle, 1990 ). Primers were designed to amplify a 560 bp fragment containing the sgRNA. The PCR products were cloned with pCR2.1®-TOPO TA vector (ThermoFisher Scientific), and 10 positive colonies were chosen and sequenced. The primers used for ptst1 amplification were 5'-TGA ATC GCG ATG TAG GTC AG-3' as forward and 5'-GCG CAC TTT GCT ATT GAA TG-3' as reverse. The primers used for gbss1a amplification were 5'-CTT CTG GCC TGC TAC CTC AA-3' as forward and 5'-TAG TTG ACG GCG AGG AAC TT-3' as reverse.
Specific primers 5'-CAG TCG CAA ATA AAG GAT GAT GC-3' as forward and 5'-CGA ACA AGA AAA TGC TAA CCT GC-3' as reverse were used to flank 15 bp deletions of PTST1 in barley T1 plants. Wild-type plants amplified 221 bp while homozygous mutant plants amplified 206 bp and heterozygous mutant plants amplified both fragments (221 bp and 206 bp).
Zhong Y., Blennow A., Kofoed-Enevoldsen O., Jiang D, & Hebelstrup K.H. (2018). Protein Targeting to Starch 1 is essential for starchy endosperm development in barley. Journal of Experimental Botany, 70(2), 485-496.
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6

TA Cloning and Sequencing of DNA

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PCR products were assembled into the pCR2.1-TOPO™ TA vector (Thermo Fisher Scientific)
using TA cloning, and the cloned vectors were transformed into DH5α competent cells
(Toyobo Co., Ltd., Osaka, Japan). Colony PCR was performed using primers M13-20
(5′-GTAAAACGACGGCCAG-3′) and M13R (5′-GGAAACAGCTATGACCATG-3′). The amplified insert DNA
was sequenced using the Big Dye Terminator Sequencing Kit (version 3.1; Applied
Biosystems, Waltham, MA, USA). The cDNA sequences were aligned with GenBank
AB618613.1.
Watanabe T., Terada T., Ezaki R., Matsuzaki M., Furusawa S, & Horiuchi H. (2024). Chicken Interleukin-5 is Expressed in Splenic Lymphocytes and Affects Antigen-Specific Antibody Production. The Journal of Poultry Science, 61, 2024002.
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7

Kmt2d cDNA Fragment Cloning and In Situ Hybridization

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A 786 bps of Kmt2d cDNA fragment was amplified using primers 5-CCGAATCAAACAGGGTCGGA −3 and 5-ACATAGGGCTGGCAGGAAAC-3 and cloned into pCR™ 2.1-TOPO® TA vector (Thermo Fisher, K450001). To generate Kmt2d antisense RNA probe, the plasmid was linearized with KpnI and was transcribed with T7 RNA polymerase. Kmt2d sense probe was generated by linearization with EcoRV and was transcribed with Sp6. In situ hybridization was performed on sections using both antisense and sense probes, with the latter serving a negative control. Experimental procedure of In situ hybridization was carried out as described previously (Yang et al., 2018 (link)).
Dong C., Umar M., Bartoletti G., Gahankari A., Fidelak L, & He F. (2019). Expression pattern of Kmt2d in murine craniofacial tissues. Gene expression patterns : GEP, 34, 119060.
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8

Detection and Sequencing of BMMF Amplicons

Cited 2 times
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After completion of RCA, all samples underwent a PCR procedure for the detection of short-size BMMF amplicons (approx. 300–600 bp). The applied primers, PCR reaction set-ups and thermal programs have been published elsewhere [35 (link),36 (link)]. In case of positive results, primer pairs for inverse PCR were designed based on the sequences of short fragments, followed by amplification of full-length sequences. Full-length amplicons were cloned into pCR2.1 TOPO TA vector (ThermoFisher Scientific Inc., Waltham, MA, USA). After transformation of E. coli, MiniPrep and restriction digest, insert-bearing clones were subjected to Sanger Sequencing on both strands. MWG Eurofins (Ebersberg, Germany) operated all sequencing reactions.
König M.T., Frölich K., Jandowsky A., Knauf-Witzens T., Langner C., Dietrich R., Märtlbauer E, & Didier A. (2023). First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle. Animals : an Open Access Journal from MDPI, 13(9), 1492.
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9

Identification and Characterization of FGFR3 Isoforms

Cited 1 time
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RNA was purified from cell lines using the QIAgen miRNeasy kit (Valencia, CA) and RT-PCR was performed to amplify FGFR3-L and FGFR3-S using random hexamers (Thermo Fisher Scientific). 5’ and 3’ rapid amplification of cDNA ends (RACE) was performed as previously described8 (link). RACE primers were designed based on the reference sequence of FGFR3 provided by the National Center for Biotechnology Information (NCBI) and are listed in Supplemental Table S1. RT-PCR products were ligated into pCR2.1-TOPO TA vector (Thermo Fisher Scientific) according to manufacturer’s instructions. Three to 4 clones per cell line were sequence verified (Sequetech, Mountain View, CA) and the consensus sequence of the full-length FGFR3-S variant was deposited in GenBank (Accession #MK542707).
Olender J., Wang B.D., Ching T., Garmire L.X., Garofano K., Ji Y., Knox T., Latham P., Nguyen K., Rhim J, & Lee N.H. (2019). A Novel FGFR3 Splice Variant Preferentially Expressed in African American Prostate Cancer Drives Aggressive Phenotypes and Docetaxel Resistance. Molecular cancer research : MCR, 17(10), 2115-2125.
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10

Sanger Sequencing of IRF4 Exon 3

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The full-length cDNA generated from the EBV-B cells of IRF4-heterozygous and WT homozygous individuals was used for the PCR amplification of exon 3 of IRF4. The products obtained were cloned with the TOPO TA cloning kit (pCR2.1-TOPO TA vector, Thermo Fisher Scientific), according to the manufacturer’s instructions. They were then used to transform chemically competent bacteria, and 100 clones per individual were Sanger-sequenced with M13 primers (forward and reverse).
Guérin A., Kerner G., Marr N., Markle J.G., Fenollar F., Wong N., Boughorbel S., Avery D.T., Ma C.S., Bougarn S., Bouaziz M., Béziat V., Della Mina E., Oleaga-Quintas C., Lazarov T., Worley L., Nguyen T., Patin E., Deswarte C., Martinez-Barricarte R., Boucherit S., Ayral X., Edouard S., Boisson-Dupuis S., Rattina V., Bigio B., Vogt G., Geissmann F., Quintana-Murci L., Chaussabel D., Tangye S.G., Raoult D., Abel L., Bustamante J, & Casanova J.L. (2018). IRF4 haploinsufficiency in a family with Whipple’s disease. eLife, 7, e32340.
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