Transcriptional start sites (TSS) were predicted using TSSPlant [26 (link)]. Experimental verification of TSS was performed with SMARTer™ RACE cDNA Amplification Kit (Clontech) using total RNA from yeast and maize (B73) respectively, which were isolated as described herein. Primers used for RACE are listed in Additional file 2 . Products were cloned into pCR™II Vector (Invitrogen) and transformed into One Shot™ TOP10 E. coli electrocompetent cells (Invitrogen), 8 colonies were sequenced.
Pcrii vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCRII vector is a plasmid-based cloning vector commonly used in molecular biology applications. It provides a stable platform for the insertion and propagation of DNA sequences of interest. The core function of the PCRII vector is to facilitate the cloning and maintenance of DNA inserts within a bacterial host.
Automatically generated - may contain errors
Lab products found in correlation
Taq polymerase, by Takara Bio (8 mentions)
Dnase 1, by Promega (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Nbt bcip, by Roche (2 mentions)
Genescan analysis software, by Thermo Fisher Scientific (2 mentions)
Bm purple, by Roche (2 mentions)
Abi3730xl, by Macrogen (2 mentions)
Abi prism model 373, by Thermo Fisher Scientific (2 mentions)
Nbt bcip ap substrate solution, by Roche (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Axyprep plasmid miniprep kit, by Corning (2 mentions)
Takara ex taq, by Takara Bio (2 mentions)
Generacer kit, by Thermo Fisher Scientific (2 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Smarter race cdna amplification kit, by Takara Bio (1 mentions)
Advantage hd polymerase, by Takara Bio (1 mentions)
Alkaline phosphatase, by Roche (1 mentions)
67 protocols using pcrii vector
1
Mapping Transcriptional Start Sites
2
Cloning and Expression of chOKT10 Antibody
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Example 5
For the construction of chOKT10 the mouse VH and VL regions were amplified by PCR using cDNA prepared from the murine OKT10 hybridoma cell line (ECACC #87021903). A set of primers was used as published (Dattamajumdar et al., 1996; Zhou et al., 1994). PCR products were used for Topo-cloning (Invitrogen; pCRII-vector) and single colonies subjected to sequence analysis (M13 reverse primer) which revealed two different kappa light chain sequences and one heavy chain sequence. According to sequence alignments (EMBL-nucleotide sequence database) and literature (Krebber et al, 1997) one of the kappa-sequence belongs to the intrinsic repertoire of the tumor cell fusion partner X63Ag8.653 and hence does not belong to OKT10 antibody. Therefore, only the new kappa sequence and the single VH-fragment was used for further cloning. Both fragments were reamplified for the addition of restriction endonuclease sites followed by cloning into the respective pMORPH® IgG1-expression vectors. The sequences for the heavy chain (SEQ ID NO: 72) and light chain (SEQ ID NO: 73) are given in
3
PCR amplification and sequencing of porcine OGT gene
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PCR reactions were carried out in a 10 μL volume containing 50 ng genomic DNA, 1.5 mM MgCl2, 10 pmol of each primer (OGT-2773F: 5′-TCTGAAGCGTGTTCCCAATAG-3′ and OGT-3083R: 5′-GCTCAAGACAACC TAAACAAGTAAG-3′), 100 μM dNTP, and 0.35 U Taq polymerase. Amplification was performed under the following PCR conditions; 10 min at 95°C; 35 cycles of 30 s at 95°C, annealing for 30 s at 61°C, 1 min at 72°C; and a final extension of 5 min at 72°C. The amplified genomic DNA in the initial study from 1 WC boar and 7 sows [2MI×WC, 2DU× WC, 2ME×WC, 1FE×WC] at USMARC were cloned into pCRII vector (Invitrogen, Carlsbad, CA, USA) and sequenced in both directions. The amplified genomic DNA of European and ME pigs at NIAS were also sequenced. Agarose gel electrophoresis and sequencing of the PCR amplicons of porcine genomic DNA indicated that the size of the amplicon from White composite and Duroc pigs were 825 bp, while those of Chinese pigs were 544 bp.
4
Catsup Transcript Amplification and Sequencing
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For each species, we prepared total RNA (Trizol, Invitrogen, Life Technologies GmbH, Germany) and synthesized cDNA (SuperScript III First-Strand Synthesis System, Invitrogen, Life Technologies GmbH, Germany) that was used to amplify Catsup transcript by PCR (Advantage HD Polymerase, Clontech) with specific primers (forward 5′-ATGGCCAAACAAGTGGCTGA-3′ and reverse 5′-TTACTCGAACTTGGCGATAAC-3′). Each PCR product was cloned in pCRII vector (Invitrogen, Life Technologies GmbH, Germany) and at least 10 colonies were picked for plasmid DNA purification and sequencing. These sequences were aligned using MegAlign (DNASTAR) and a consensus sequence was obtained for each species. The sequence of CatsupIn270Del was obtained from the Drosophila Polymorphism Database (DPDB) (Casillas et al., 2005 (link)).
5
In Situ Hybridization for QRFP Detection
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In situ hybridization was performed as previously described (Mieda et al., 2006). Digoxigenin (DIG)-labeled riboprobes for QRFP were synthesized by RT-PCR from a 0.60-kb fragment of mouse cDNA encoding prepro-QRFP from mouse brain RNA and subcloned into pCRII vector (Invitrogen). The following primers were used to amplify cDNA fragments: prepro-QRFP, 5'-CCTCCCACAGGGAGCACACCG-3' and 5'-CCTGTTGTATCCACGGCCCCA-3'. The DIG-labeled probes were detected by anti-DIG (1/1000) antibodies conjugated with alkaline phosphatase (Roche Diagnostics, Basel, Switzerland). alkaline phosphatase activity was detected with NBT/BCIP (Roche Diagnostics).
6
HO-1 Gene GT-Repeat Genotyping
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The 5′-flanking region of the HO-1 gene containing GT-repeats was amplified by polymerase chain reaction (PCR) with a FAM-labeled sense primer (5′-AGAGCCTGCAGCTTCTCAGA-3′ ) and an antisense primer (5′-ACAAAGTCTGGCCATAGGAC-3′ ) according to a published protocol [7] (link). The PCR products were mixed with the GenoType TAMRA DNA Ladder (size range: 50–500 bp; Invitrogen, Grand Island, NY) and analyzed in an automated DNA sequencer (ABI Prism 377, Foster City, CA). The respective sizes of the GT-repeats were calculated using GeneScan Analysis software (PE Applied Biosystems, Foster City, CA). To further confirm the sizes of the GT-repeats, 3 PCR products were subcloned into the pCRII vector (Invitrogen) and subjected to the sequence analysis. For quality control purposes, approximately 10% of the samples were re-genotyped in a blinded fashion, and from which the same results were obtained.
7
Panarthropod Fox Gene Identification
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For all species, RNA isolation of a mix of embryos representing different developmental stages, and subsequent cDNA synthesis were carried out as described in [34 (link)]. All gene fragments were amplified using gene specific primers (S1 Table ) based on published genomes and transcriptomes, and Topo-TA cloned into the pCRII vector (Invitrogen, Carlsbad, CA, USA). Sequences were checked on an ABI3730XL analyser using Big Dye dye-terminators by a commercial sequencing service (Macrogen, Korea). Sequences identifiers of all investigated panarthropod Fox genes are listed in S2 Table .
8
GST Pull-Down Assay Protocol
9
Endometrial and Conceptus RNA Extraction and Analysis
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Total RNA was extracted from endometrial and conceptus tissues using TRIzol reagent (Invitrogen Life Technology, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of the RNA was validated following electrophoresis in 1% agarose gel. Four micrograms of total RNA from endometrial, conceptus, and chorioallantoic tissues were treated with DNase I (Promega, Madison, WI, USA) and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen, USA) to obtain cDNA. The cDNA templates were then diluted 1:4 with sterile water and amplified by PCR using Taq polymerase (Takara Bio, Shiga, Japan). The final PCR reaction volume of 50 μL contained 3 μL of cDNA, 5 μL of 10× PCR buffer, 4 μL of dNTP mix (2.5 mM), 1 μL of each primer (20 μM), 0.3 μL of Taq polymerase (Takara Bio, Japan), and 36.7 μL of ddH2O. The PCR conditions, sequences of primer pairs for PI3 and SLPI, and expected product sizes are listed in Table 1 . The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining. The identity of each amplified PCR product was verified by sequence analysis after cloning into the pCRII vector (Invitrogen, USA).
10
Gene Expression Profiling in Tribolium
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Gene fragments were amplified by means of RT-PCR from cDNA synthesized from either total RNA or messenger RNA. Gene-specific primers were designed based on available sequence information (Tribolium Genome Sequencing Consortium [26 (link), 35 (link), 60 , 61 (link)] (Additional file 1 : Table S1)).
All amplified gene fragments were cloned into the PCRII vector (Invitrogen). Sequences of the cloned fragments were sequenced on an ABI3730XL automatic sequencer (Macrogen, Seoul, South Korea). Gene fragment identification numbers are summarized in Additional file2 : Table S2.
The whole-mount in situ hybridization protocol was used as described in [62 (link)]; for confocal microscopy, we stained embryos with SIGMAFAST Fast Red TR/Naphtol AS-MX (SIGMA) instead of BM Purple (ROCHE). Cell nuclei were visualized incubating embryos in 5 μg/ml of the fluorescent dye 4-6-diamidino-2-phenylindole (DAPI) in phosphate buffered saline with 0.1% Tween-20 (PBST) for 20 min, followed by several washes in PBST to remove excess DAPI.
All amplified gene fragments were cloned into the PCRII vector (Invitrogen). Sequences of the cloned fragments were sequenced on an ABI3730XL automatic sequencer (Macrogen, Seoul, South Korea). Gene fragment identification numbers are summarized in Additional file
The whole-mount in situ hybridization protocol was used as described in [62 (link)]; for confocal microscopy, we stained embryos with SIGMAFAST Fast Red TR/Naphtol AS-MX (SIGMA) instead of BM Purple (ROCHE). Cell nuclei were visualized incubating embryos in 5 μg/ml of the fluorescent dye 4-6-diamidino-2-phenylindole (DAPI) in phosphate buffered saline with 0.1% Tween-20 (PBST) for 20 min, followed by several washes in PBST to remove excess DAPI.
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