100ng-150ng of DNA was amplified with primers “FMR1 F_up” and “FMR1 R_down” (final concentration: 500nM, Table 1 ) using the Expand Long Template PCR System (Sigma)56 (link). Reactions took place in Buffer 2, and were supplemented with betaine (final concentration: 1.2M) and DMSO (5% v/v). Cycling conditions were programmed as in Saluto, et al., 2005.
Expand long template pcr system
Manufactured by Merck Group
Sourced in United States
The Expand Long Template PCR System is a laboratory equipment designed for amplifying long DNA fragments using a robust and efficient PCR (Polymerase Chain Reaction) process. The system combines a specialized enzyme mix and optimized reaction buffer to enable the amplification of DNA targets up to 40 kilobases in length.
Automatically generated - may contain errors
Lab products found in correlation
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Kapa hifi 2x readymix, by Roche (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Monarch plasmid miniprep kit, by New England Biolabs (1 mentions)
6 protocols using expand long template pcr system
1
FMR1 Gene Amplification for Genetic Analysis
2
Cloning and Expressing pqsL in P. aeruginosa
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pqsL was amplified with the Expand long-template PCR system (Sigma) from P. aeruginosa PA14 chromosomal DNA using the forward primer 5′-GAATTCGGAACGACACGGAGACTCATCC -3′ and reverse primer 5′-GAGCTCAGCCGCGCGGAGC -3′. The 1,238-bp amplicon was ligated into the TOPO cloning vector using the TOPO TA cloning kit (Thermo Fisher) to create pTOPO-pqsL. pqsL was then removed from pTOPO-pqsL by EcoRI digestion and cloned into EcoRI-digested pBBR1MSC-5 (63 (link)). In the resulting plasmid (pBBR1-pqsL), pqsL is oriented such that it is transcribed from the lac promoter.
3
Isolation and Sequencing of Wx Gene
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Genomic DNA was isolated from young seedlings, as described.6) (link) The oligonucleotide sequences for amplification and sequencing are summarized in Supplemental Table 1 and Supplemental Fig. 1 . PCR conditions for amplification of the Wx gene were 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 3 min using the Expand Long Template PCR system (Sigma Aldrich, St. Louis, MO, USA former Roche, Basel, Switzerland) supplemented with 2.5 % dimethyl sulfoxide. PCR products were separated by 0.8 % agarose, and the corresponding band was excised and purified using a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands). Purified DNA was sequenced with listed primers (Supplemental Table 1 and Supplemental Fig. 1 ) using BigDye Terminator (Thermo Fisher Scientific Inc., Waltham, MA, USA former Applied Biosystems, Foster city, CA, USA) at the Biotechnology Center in Akita Prefectural University.
4
FMR1 Gene Amplification for Genetic Analysis
5
Efficient DNA Extraction and Amplification
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Plasmid DNA was prepared using a Monarch Plasmid Miniprep Kit (NEB, cat# T1010). Genomic DNA was prepared using the DNeasy Blood & Tissue Kit (Qiagen, cat# 69504). When necessary, cDNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB, cat# T1030). PCR was performed using either KAPA HIFI 2X ready mix (KAPA Biosystem, cat# KK2602) or Expand Long Template PCR System (Sigma, cat# 11681842001). Primers used for each PCR reaction are listed in S4 Table .
6
Quantification of Mitochondrial DNA
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Frozen muscle samples were homogenized with a handheld homogenizer in very short bursts, and total DNA was purified using phenol-chloroform extraction. The mtDNA copy number was quantitated by quantitative PCR (qPCR) using primers specific to mouse mtDNA and nuclear DNA (nDNA), and the ratio was calculated using the 2−ΔΔCT method (51 (link)). Long-range PCR over the mitochondrial genome was performed using the Expand Long Template PCR system (Sigma-Aldrich, 11681834001), following the manufacturer’s instructions. Samples were visualized on a 0.5% agarose gel.
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