Short fragments of CoI/II and EF1α were sequenced from historical specimens up to 150 years of age, obtained from museum and private collections (online Appendix S1, available on Dryad at http://dx.doi.org/10.5061/dryad.44b4j ) and processed in a vertebrate genetics laboratory to reduce the risk of contamination. Instruments and surfaces were cleaned with 5% bleach and irradiated with UV for 30 min prior to use. One to two legs were washed in water, immersed in liquid nitrogen in a test tube for 30 s and ground up, followed by an extraction into 20 μl of buffer using the QIAmp DNA Micro Kit (Qiagen, Manchester, UK). We treated every fifth extraction and every fifth PCR as a negative control with no tissue or DNA extract. PCRs were carried out in a 20-μl volume using 1 unit of Platinum HiFi Taq Polymerase (Invitrogen, London, UK) and 1× buffer, 2.5 mM MgCl2, 0.5 μM of each primer, 0.2 mM dNTPs, 1 unit bovine serum albumin, sterilized DNAse-free water and 1–5 μl of the DNA extract depending on concentration. To accommodate shearing of DNA with time, we designed and applied PCR primers spanning short fragments of 200–300 bp (online Appendix S2, available on Dryad at http://dx.doi.org/10.5061/dryad.44b4j ). We carried out amplification, product clean up and sequencing as above, partially accounting for possible cross-contamination by blasting the results against GenBank.
Platinum hi fi taq polymerase
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Platinum Hi-Fi Taq polymerase is a high-fidelity DNA polymerase used for PCR amplification. It exhibits a proofreading activity, resulting in increased accuracy during DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Qiamp dna micro kit, by Qiagen (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Bl21 de3 plys, by Thermo Fisher Scientific (1 mentions)
Xhoi, by Takara Bio (1 mentions)
Avrii, by Takara Bio (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Lr clonase reaction, by Thermo Fisher Scientific (1 mentions)
5 protocols using platinum hi fi taq polymerase
1
Genetic Analysis of Historical Specimens
2
Competent Cell Expression System
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DH5a competent, BL21 (DE3) pLysS, and Rosetta cells were purchased from Invitrogen (Carlsbad, CA, USA). The pMD-18T vector was purchased from TaKaRa Biotechnology (Dalian, China). The pET22B, pPIC9, and pTT5 vectors, HEK293 cells, and Pichia GSll5 were stored in our laboratory. Polymerase chain reaction (PCR) products were extracted using a gel extraction kit (BBI, Canada). Restriction enzymes (NcoI, XhoI, AvrII, and NotI) and T4 DNA ligase were purchased from TaKaRa Biotechnology. Platimum Pfx Polymerase and platinum HIFI Taq Polymerase were purchased from Invitrogen. The primers were synthesized by Shanghai Biological Engineering Technology (Shanghai, China). PCR products were sequenced using the ABI PRISM 377 DNA auto sequencer (Applied Biosystems, Foster City, CA, USA).
3
Fusion of lis1 Genes with Fluorescent Tags
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Standard PCR and cloning procedures (Sambrook et al., 1989) were used to fuse the sgfp gene to the end of the lis1-1 and lis1-2 structural genes. The lis1-1 and lis1-2 genes were amplified by PCR from N. crassa (FGSC 2489) genomic DNA. Primers used are listed in Table 1. PCR was performed in an Apollo Thermal Cycler with Platinum Hi-fi Taq polymerase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Amplified and gel-purified PCR products were digested with XbaI and PacI and ligated into XbaI-and PacI-digested plasmid pMF272 (GenBank accession No. AY598428) for GFP tagging and pJV15-2 for mChFP (Verdín et al., 2009) . The resulting expression plasmids were pRM04-OC03, pRM13-OC06, pRM23-OC16 and pRM22-OC15 containing the gene fusions lis1-1-sgfp, lis1-2-sgfp, lis1-1-mChPF and lis1-2-mChPF, respectively (Table 1). All plasmids were verified by sequencing at Eton Biosciences (San Diego, CA).
4
Cloning and Expression of CYP72A Genes
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Coding sequences information for MtCYP72A67 and MtCYP72A68 (Li et al. 2007) (link) were obtained from NCBI genebank: DQ335782 and DQ335780, respectively. Primers were designed for the coding sequence by using Primer3 software (Rozen and Skaletsky 2000) . The upstream cloning primer for MtCYP72A67 and MtCYP72A68 included both a BamHI restriction site and a kozak yeast translation initiation sequence, where the downstream cloning primer included an EcoRI cut site. MtCYP72A67 and MtCYP72A68 were amplified from cv. Jemalong A17 aerial tissue cDNA template using Platinum Hi-Fi Taq polymerase (Invitrogen, Carlsbad, CA). The CYP72A68 PCR product was cloned into the pGEM-T easy vector (Promega, WI), then sequenced using M13 forward and reverse primers. CYP72A67 and CYP72A68 were excised from the p-GEM easy vector via a BamHI and EcoRI restriction digest and sub-cloned into the pYeDP60 vector (Pompon et al. 1996) (link) and sequenced using the GAL10 promoter. WAT11 yeast cells were transformed as previously reported (Greenhagen et al. 2003; (link)Urban et al. 1997 ) and confirmed by PCR.
5
Overexpression and RNAi of CYP72A genes
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The coding sequence of MtCYP72A67 and MtCYP72A68 were amplified from cDNA synthesized from M. truncatula (cv. Jemalong A17) aerial tissue using Platinum Hi-Fi Taq polymerase (Invitrogen, Carlsbad, CA). The primer sequences used for amplification are listed in Sup. Table S9. PCR products were cloned into the entry vector pENTR/D/TOPO (Invitrogen) and sequenced. The entry vectors were recombined into a destination vector, pK7WG2D for overexpression or pK7GWIWG2D(II) for RNAi (a double-stranded hairpin RNA), by using the LR clonase reaction (Invitrogen). The vectors were transformed into Agrobacterium rhizogenes (strain ARqua1) by electroporation (Quandt et al. 1993) (link). Transformed colonies were grown on LB-agar medium at 28°C, with spectinomycin and streptomycin for vector selection. After confirmation by PCR, transformed agrobacteria were used to transform leaves of M. truncatula (cv. Jemalong A17) and generate hairy roots (Verdier et al. 2012) (link).
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