PCR amplification of the 455 bp fragment of the 16 S rDNA gene was performed using a single pair of primers 16 S-F: TTA AAT TGC TGT RGT ATT and 16 S-R1: CCG GTC TGA ACT CASAWC and thermo cycling conditions as previously described [17 (link)]. Each reaction was prepared in a final volume of 25 µl containing; − 10 µl of 1× PCR buffer, 20 µM of each dNTPs, 1.5 mM MgCl2, 0.25U of Taq Polymerase, 1 µl of 0.2 µM of each primer, 7 µl of nuclease free water, and 5 µl (containing 1ng – 1ug) of genomic DNA template. The amplification was completed in a thermocycler (MultiGene OptiMax Thermal Cycler, Labnet International, Edison, NJ, USA) with initial denaturation of 94° C for 5 min, followed by 35 cycles at 94° C for 30 s, 48° C for 45 s, 72° C for 45 s, and a final extension at 72° C for 7 min. Five microliters of each PCR amplicon stained with ethidium bromide were run on 2% agarose gels and visualised on an ultraviolet transilluminator to check the quality and yield of PCR product. The resultant PCR products were sized against a 1 kb DNA molecular ladder (Bioline, London, UK). PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and commercially Sanger-sequenced (Inqaba Biotec, Pretoria, South Africa).
1 kb dna molecular ladder
Manufactured by Meridian Bioscience
Sourced in United Kingdom
The 1 kb DNA molecular ladder is a laboratory tool used to determine the size of DNA fragments during gel electrophoresis. It consists of a set of DNA fragments of known molecular weights, typically ranging from 250 to 10,000 base pairs, which can be used as a reference to estimate the sizes of unknown DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using 1 kb dna molecular ladder
1
16S rDNA PCR Amplification and Sequencing
2
Molecular Identification of Organisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR ampli cation was performed on 12S rRNA and 16S rRNA genes and the ITS 2 spacer using primers (Table 1) and thermocycling conditions as previously described [4, 19] . Each reaction was prepared into a nal volume of 50 µl containing; 1´-reaction buffer (670 mM Tris-HC , pH 8.8, 166 μM (NH 4 ) 2 SO 4 , 4.5 % Triton X-100, 2 mg/ml gelatin) (Bioline, Humber Road, London, UK), 0.25 mM of each dNTP, 0.25 mM each of forward and reverse primers, 1.56 U BioTaq DNA polymerase (Bioline, London, UK), 1.25 mM MgCl 2 , 32.2 µl of PCR grade water and nally 5 µl of the template DNA.
The 16S ribosomal RNA gene was ampli ed in a thermocycler (Personal Thermocycler, Biometra, Göttingen, Germany) with initial denaturation of 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 48 °C for 45 s, 72 °C for 45 s and a nal extension at 72 °C for 7 min. Ampli cation of the ITS2 and 12S ribosomal RNA was performed using similar thermocycling conditions to those of 16S at annealing temperature of 55 °C and 52 °C, respectively. PCR products were resolved on 2% agarose gels. The resultant PCR products were sized against a 1 kb DNA molecular ladder (Bioline, London, UK). The expected PCR product sizes ranged between 300-1200 bp. PCR products were puri ed using QIAquick PCR Puri cation Kit (Qiagen, Germantown, MD, USA) and commercially Sanger-sequenced (Inqaba Biotec, Muckleneuk, Pretoria, South Africa).
The 16S ribosomal RNA gene was ampli ed in a thermocycler (Personal Thermocycler, Biometra, Göttingen, Germany) with initial denaturation of 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 48 °C for 45 s, 72 °C for 45 s and a nal extension at 72 °C for 7 min. Ampli cation of the ITS2 and 12S ribosomal RNA was performed using similar thermocycling conditions to those of 16S at annealing temperature of 55 °C and 52 °C, respectively. PCR products were resolved on 2% agarose gels. The resultant PCR products were sized against a 1 kb DNA molecular ladder (Bioline, London, UK). The expected PCR product sizes ranged between 300-1200 bp. PCR products were puri ed using QIAquick PCR Puri cation Kit (Qiagen, Germantown, MD, USA) and commercially Sanger-sequenced (Inqaba Biotec, Muckleneuk, Pretoria, South Africa).
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!