Pcr4 topo

Total RNA preparations, reverse transcription (RT) and quantitative PCR (qPCR) reactions were performed as previously described [41 (link)]. qPCR standards consisted of 10-fold dilutions of quantified plasmids carrying the sequence to be amplified: pTM793 for β-actin, pMK72 for Apol9b, pMK68 for Apol9a and pCS40 for Oasl2. These plasmids are derivatives of pCR4-ToPo (Invitrogen) in which the corresponding PCR fragments were cloned. Real-time PCR primer sequences and reference plasmid used are given in Table 3.

B. thuringiensis subsp. kurstaki HD1 (Bt HD1), B. thuringiensis subsp. kurstaki HD73 (Bt HD‐73), and B. thuringiensis subsp. tenebrionis (Btt) DSM‐2803 are sporogenic bacteria kindly provided by Jorge Ibarra (CINVESTAV Irapuato, Mexico). The chitinase gene (chiA Btt) was cloned from B. thuringiensis subsp. tenebrionis DSM‐2803, a bacterium that is toxic to coleopteran larvae owing to its quadrangular‐flat crystal composed of Cry3 protein protoxins (~74 kDa). Recombinant plasmids were propagated in Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) and E. coli BL21 Rosetta 2 (Merck Millipore, MA) for the purpose of cloning the gene, and for production and purification of the enzyme, respectively. Plasmids pCR 4‐TOPO (Invitrogen), pCold I (Takara Bio Inc, Otsu, Shiga, Japan), or pHT3101 (Barboza‐Corona et al. 2009) were used as cloning vectors. The first two plasmids are able to replicate in E. coli, whereas the pHT3101 is a shuttle vector with two replication origins, one for E. coli and the other for B. thuringiensis.

The FGSG_02809, FGSG_02810, and FGSG_02811 genes, including regions upstream and downstream of the open reading frame, were amplified from Fg0407011 by PCR using the HS458/HS459, HS450/HS451, and HS442/HS443 primer pairs, respectively (Table S1). PCR products treated with NotI were inserted into the NotI site in pCB1004 to create the respective transformation vectors pCB02809, pCB02810, and pCB02811 using DNA Ligation Kit Ver. 2 (Takara, Otsu, Japan).
The FGSG_02810 replacement vector pCR402810dis was constructed as follows. The PCR product from FGSG_02810 without NotI treatment was cloned into pCR4TOPO (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions to obtain plasmid pCR402810. The hygromycin-resistance cassette region (TrpC promoter and hygromycin phosphotransferase coding sequence) in pCB1003 (Sweigard et al. 1997 ) was amplified by PCR using primers HS708 and HS709 in which XhoI and SacII recognition sequences were integrated, respectively (Table S1), using pCB1003 as the template DNA.
PCR was performed by using AmpliTaq DNA polymerase (Life Technologies), and the following cycling parameters: 94° for 2 min, 30 cycles of 94° for 1 min, 60° for 1 min, and 72° for 1 min. Amplicons treated with XhoI and SacII were inserted into XhoI and SacII sites in pCR402810 (pCR402810dis) using DNA Ligation Kit Ver. 2.

Mouse embryos used in whole mount in situ hybridizations and gonad cultures were obtained from matings between CD1 random bred mice (Charles River Labs). Noon of the day when vaginal plug was recorded was considered E0.5. Whole mount in situ hybridizations with the Stra8 probe were performed as previously described [3] (link), [39] (link). Digoxigenin riboprobe for Rec8 was generated by amplifying cDNA fragments by RT-PCR from Rec8 (NM_020002.2: bases 274–865), and inserting them into TA cloning vector pCR4-TOPO (Invitrogen). Plasmid was linearized with Spe1 or Not1 and transcribed with T7 or T3 respectively to make the antisense and sense probes.

Total RNA from ovules of flowers at stage 11 from two haploid-producing 4x T1 progeny (K6.1_20 and K6.1_23) of line RKD2gBB_6.1 was extracted, DNase treated and converted into single strand cDNA as previously stated. PCR reactions consisted of 2 µL of the first-strand cDNA synthesis reaction, 1× PrimeSTAR GXL Buffer, 200 µM dNTP, 0.2 µM primers, and 0.625 U PrimeSTAR GXL DNA Polymerase in a 25 µL reaction (Takara Bio USA, Inc) followed by 35 cycles of amplification with a cycling condition of 15 s at 98 °C, 15 s at 60 °C, and 2 min at 68 °C. Primers p1792/1801 (Table S1) were used to amplify the transcript from the start of the ORF through to the 3′ UTR. Amplified PCR products from each sample were directly cloned into PCR4-TOPO (Invitrogen) vector with cloned inserts sequenced at Psomagen (Rockville, MD, USA). Sequencing data were analyzed using Geneious prime software (Biomatters Limited, Auckland, New Zealand).