Pgem t easy vector

For ancient samples a 532 base pair subsection from the 5′ end of the mitochondrial control region (CR) was PCR amplified using a combination of the overlapping primers listed in Table S2. PCR Reactions (25 µl) contained 2 µl of DNA extract, 22.6 µl Multiplex PCR Kit (Qiagen) and 0.2 µM of each primer. Thermal cycling conditions were as follows: an initial denaturation of 15 min at 95 °C followed by 45 cycles of 30 s at 95 °C, 90 s annealing, 45 s at 72 °C followed by a final extension for 30 min at 60 °C. Negative controls (no DNA) were used for all PCR runs. Column-purified PCR products were Sanger sequenced at DBS Genomics, Durham University. Samples showing any sequencing ambiguities were replicated for all steps.
To help assess and quantify post-mortem damage and exogenous DNA content, for 6 ancient samples the PCR product was cloned into the pGEM-T Easy vector (Invitrogen) and transformed into JM109 competent cells (by heat-shock for 45–50 s). After blue/ white selection, positive colonies were miniprepped and the insert DNA amplified by PCR using T7 and Sp6 primers. A minimum of 6 clones were sequenced on an ABI automated sequencer from all 6 samples (chosen at random) to check for potential contamination or incorrect base calls. During cloning and sequencing, no evidence of nuclear copies was detected.

The activation-tag insertion site in mutant A2 was determined using thermal asymmetric interlaced PCR (TAIL-PCR) [46] (link). The PCR was performed with a combination of nested primers [47] (link) and 10-mer random primers [48] (link). The secondary and tertiary TAIL-PCRs were separated on 1.2% agarose gel, stained with ethidium bromide, and visualized using the ChemiDoc XRS system (Bio-Rad). Specific product, judged based on the size differences generated by the nested primers, was excised, cleaned using the QIAquick Gel Extraction Kit (QIAGEN), cloned into the pGEM-T Easy Vector (Invitrogen), and sequenced. Blastn search of the TAIR database using the PCR sequences was performed to identify the genomic insertion site. Based on the putative insertion site, the primer pair MPR15F and MPR15R were designed and used to amplify the flanking genomic region. By sequencing this region in the wild-type and the mutant A2, the exact insertion site was determined.

qPCR was used to validate the aCGH data. Primer amplification efficiency for each gene was determined. Specific sequences of the reference and target genes were cloned into pGEM-T easy vector (Invitrogen). Known amounts of genomic DNA (from clones D11 and CLB and G strain) and recombinant plasmids containing the sequences of interest were incubated with 10 µL SYBR Green-Based Detection (Applied Biosystems), sense and antisense primers and water to a total reaction volume of 20 µL. The reaction mixture was distributed into 0.2 mL tubes and subjected to 40 cycles of amplification in the Rotor-Gene^® Q PCR cycler (Qiagen) according to the manufacturer’s instructions. The qPCR program was set as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 60 s. The results were analyzed with Rotor-Gene 6000 v1.7 software (Qiagen). A standard curve was constructed for each target gene. Data obtained by amplification with genomic DNA samples could be compared as the amount of genomic DNA was the same for the three T. cruzi isolates. To estimate the copy numbers of each target gene in the three isolates, data were normalized separately using the genome size of each T. cruzi isolate (Souza et al., 2011 (link)). All experiments were performed in triplicate.

Genomic DNA was isolated from fresh leaves using the GeneElute Plant Genomic Miniprep Kit (Sigma).
PCR amplifications were carried out using 80 ng of genomic DNA, 5 μl 10X Buffer, 1 μl 10 mM dNTPs, 2 μl 10 μM forward and reverse primers, 2U of Triple-Master Polymerase Mix (Eppendorf). After the first DNA denaturation step at 94°C for 3 min, amplifications were run for 30 cycles consisting of 1 min at 94°C, 1 min at 69°C, and 1 min at 72°C. A final elongation step was then run at 72°C for 7 min. PCR products were analyzed on 1.5% agarose gel and purified with the PCR product purification kit “GFX^TM PCR DNA and Gel Band Purification” Kit (GE Healthcare). The purified amplifications were then cloned using the pGem-T Easy vector (Invitrogen) and transformed into JM109 competent Escherichia coli cells by the heat shock method. Positive clones were selected and their plasmids were sequenced at both insert ends with SP6 and T7 primers. The full fragment sequence was obtained by primer walking using specific primers (Supplementary Table S1). Regions of ambiguous reads were re-sequenced with additional primers.

The Amt coding region was amplified from CS antennal cDNA and cloned into the pGEM-T Easy vector (Invitrogen) for transcription. Digoxigenin (DIG) labeled probes for Amt were created using the DIG RNA Labeling Kit (SP6/T7) (Roche) and hydrolyzed for 1 hour in 30 mM Na₂CO₃, 20 mM NaHCO₃ (pH 10.2). The reaction was stopped with 3 M NaOAc, 1% acetic acid (pH6), and then the probe was purified via ethanol precipitation, solubilized in DEPC H₂O, and stored at -80°C. The mCD8::GFP probe was created similarly but from the pBS mCD8::GFP plasmid [103] (link) using T3 polymerase and Fluorescein (FITC) labeled UTP (Roche). The Obp84a FITC probe was created similarly, but was not hydrolyzed, and was instead purified with the RNEasy Cleanup Kit (QIAGEN).

The coding region of each Obp was amplified from CS antennal cDNA and cloned into the pGEM-T Easy vector (Invitrogen, Waltham, MA) for transcription. For genes with multiple transcripts, the primers were designed to encompass the region present in all versions. Plasmids were linearized with SpeI, NotI, or AatII (New England BioLabs). Digoxigenin (DIG) and Fluorescein (FITC) labeled probes were created using DIG RNA Labeling Kit SP6/T7 and Fluorescein-labeled UTP (Roche, Branford, CT), and purified with the RNEasy Cleanup Kit (QIAGEN, Germantown, MD).