Common molecular biology procedures including plasmid DNA transformation, restriction and amplification were performed as previously described by Sambrook et al. [31 ]. All modification enzymes and restriction endonucleases were used according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Mini- and midi-scale plasmid preparations were obtained using the NucleoSpin plasmid and the NucleoBond Xtra Midi plasmid purification kits, respectively (Macherey-Nagel GmbH & Co., KG, Düren, Germany). Following PCR amplification, DNA fragments were purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, KG, Düren, Germany). PCR reaction was carried out in an Applied Biosystems SimpliAmp thermal cycler (Thermo Scientific, Waltham, MA, USA) using PCRBIO Classic Taq and PCRBIO HiFi polymerase (PCR Biosystems Ltd., London, United Kingdom) on C. jejuni NCTC 11168 genomic DNA template. All plasmids and primers used in this work are listed in Table S1 .
Pcrbio classic taq
Manufactured by PCR Biosystems
Sourced in United States
PCRBIO Classic Taq is a DNA polymerase enzyme suitable for standard PCR applications. It provides reliable and consistent amplification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin plasmid, by Macherey-Nagel (1 mentions)
Nucleobond xtra midi plasmid purification kit, by Macherey-Nagel (1 mentions)
Pcrbio hifi polymerase, by PCR Biosystems (1 mentions)
Ptc 100, by Bio-Rad (1 mentions)
Hifi polymerase, by PCR Biosystems (1 mentions)
Script cdna synthesis kit, by Jena Biosciences (1 mentions)
Life technology, by Thermo Fisher Scientific (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
3 protocols using pcrbio classic taq
1
Standard Molecular Biology Procedures
2
Plasmid DNA Preparation and Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA manipulations were performed as described previously [37 ]. All restriction and modification enzymes were used according to the manufacturers’ instructions (New England Biolabs, Ipswich, MA, USA). Plasmid DNA preparations were carried out with the NucleoBond Xtra Mini/Midi plasmid purification kits (Macherey-Nagel GmbH & Co, Düren, Germany). DNA fragments for cloning purposes were extracted and purified from agarose gel using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel GmbH & Co., Düren, Germany). PCRs were carried out in a PTC-100 (MJ Research, St. Bruno, QC Canada) or in an AimpliAmp (Applied Biosystems, Waltham, MA, USA) thermal cycler using PCRBIO Classic Taq or HiFi polymerase (PCR Biosystems, London, UK).
3
Semiquantitative RT-PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the cells using PureLink ® RNA Mini Kit and the elimination of any genomic DNA was performed by on-column DNAse treatment (Life technology; Thermo Fisher Scientific, Inc., Walthan, MA, USA). RNA concentration was evaluated by spectrophotometric reading at 280 and 260 nm. Total RNA was used for first strand cDNA synthesis with SCRIPT cDNA Synthesis Kit and Oligo-dT, as random primer (Jena Bioscience GmbH, Jena, Germany). The PCR was performed with about 150 ng cDNA using PCRBio Classic Taq (PCR Biosystems Ltd; London Bioscience Innovation Centre, London, NW1 ONH, United Kingdom). Experimental protocols for semiquantitative PCR reactions were: Denaturation, 95˚C for 3 min; followed by 40 cycles of denaturation at 95˚C for 15 sec. Sequences of primers and temperature of annealing used for PCR analysis are reported in Table I. PCR products were then analysed by 1.5% agarose gels electrophoresis in TBE 1X Buffer. Image acquisition and product analysis was made by Bio-Rad imaging systems with Quantity One ® 1-D analysis software. The density of the PCR bands were divided by that of the housekeeping gene (GAPDH) and expressed as a percentage of the control band density.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!