PCR products were purified either using the innuPREP DOUBLEpure Kit (Analytikjena) or the QIAquick PCR Purification Kit Protocol (QIAGEN) following the manufacturer’s instructions. Purified PCRs were sent for sequencing to Eurofins Genomics (Ebersberg, Germany), the allelic distribution profiles were determined using the online toolkit TIDE with standard parameters [8 (link)].
Qiaquick pcr purification kit protocol
Manufactured by Qiagen
Sourced in Germany
The QIAquick PCR Purification Kit is a laboratory equipment product that provides a simple and efficient method for purifying PCR amplification products. It is designed to remove unwanted primers, nucleotides, enzymes, and other impurities from PCR reactions, allowing for the subsequent use of the purified DNA in various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
3500 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Innuprep doublepure kit, by Analytik Jena (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using qiaquick pcr purification kit protocol
1
PCR Purification and Sequencing
2
Cloning ACVRL1 Gene Fragments
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We used pSpliceExpress vector, a generous gift from Dr S. Stamm (University of Kentucky, USA). Three mutations were analysed in this study: the missense mutations c.733A>G (p.Ile245Val) in exon 6, c.1249A>T (p.Ile417Phe) in exon 9, and the intronic mutation c.1048+5G>A in intron 7. Patient DNA carrying each mutation was used to generate genomic fragments of ACVRL1 including exon 6, exon 9 and exon 7 respectively with ±200bp of its upstream and downstream intronic sequences. These fragments were amplified by standard PCR using specific primers that contain the attB1 adapter F and attB2 adapter R recombination sites (listed in S2 Table ), and Platinum Taq DNA Polymerase High Fidelity (Invitrogen). PCR products were then purified using QIAquick PCR Purification Kit Protocol (QIAGEN). Next, 5–150 ng of the attB-PCR product was mixed to 150 ng of pSpliceExpress vector in 10 μl of recombination reaction, to which was added 2 μl of the BP Clonase II enzyme mix and sufficient volume of TE buffer, pH 8, to complete the total reaction volume. The reaction was incubated at 25°C for 1 hour. Then 1 μl of proteinase K (2 mg/ml; Invitrogen) was added to inactivate the enzymes. The samples were subsequently incubated at 37°C for 10 minutes to terminate the reaction and used later to transform competent E. coli (Invitrogen).
3
16S rDNA Amplification and Purification
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The 16S rDNA colony PCR products were clean-up using QIAquick PCR purification kit/protocol (Qiagen, UK). The clean products was quantified using Cubit 4 Fluorometer (ThermoFisher Scientific, UK). DNA purity was estimated by Lambda XLS UV Spectrophotometer, L7110191 (Perkin-Elmer Inc., USA). UV absorbance at 260, 280, and 230 was measured according to manufacturers' instructions. The absorbance at 260 nm (A260) and at 280 nm (A280) for the DNA was measured to determine its purity. The quantified DNA was finally analyzed by sequencing techniques for the species identification at the Medical Research Institute, University of Dundee, UK.
4
Nucleotide Sequencing Protocol Optimization
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In preparation for the nucleotide sequencing process, the amplified product was purified using the QIAquick PCR purification kit protocol (Qiagen, Germany). The nucleotide sequencing processes were as follows: The second PCR (cycling sequence) was performed using the Big Dye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, USA). The second PCR reaction mixture consisted of 8 μl of Big Dye Terminator, 3.2 μl (1 pmol) of primer (forward or reverse), 2 μl (10 ng/ μl) of PCR product and adjusted to 20 μl with nuclease-free water. The sequencing PCR reaction was carried out at 96 °C for 2 min, followed by 25 cycles of 10s at 96 °C, 5s at 51 °C and 4 min at 60 °C. The product then was purified with CENTRI-SEP columns (Princeton Separation). 10 μl of Hi-Di formamide was added to the purified product before obtained in DNA sequencer. The DNA sequencing was applied by 3500 genetic analyzer (Applied Biosystems, USA).
5
PCR Purification and DNA Sequencing
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Two PCR products were purified using QIAquick PCR purification kit protocol (Qiagen, Germany), followed DNA sequencing processes; second PCR (cycling sequence) was carried out using the Big Dye Terminator version. 3.1 Cycle Sequencing kit (Applied Bosystems, USA). Second PCR reaction mixture consisted of 8 μl of Big Dye Terminator, 3.2 μl (1 pmol) of primer (forward or reverse), 2 μl (10 ng/ μl) of PCR product and completed to 20 μl with nuclease-free water. The sequencing PCR reaction was carried out at 96 °C for 2 min, followed by 25 cycles of 10 s at 96 °C, 5 s at 51 °C and 4 min at 60 °C. The product then was purified with CENTRI-SEP columns (Princeton Separation). 10 μl of Hi-Di formamide was added to the purified product before introduced in DNA sequencer. DNA sequencing was applied by 3500 genetic analyzer (Applied Biosystems, USA).
6
16S rRNA Gene Amplification and Purification
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All out genomic DNA was separated from unadulterated societies utilizing QIAamp PCR Kit (Qiagen, Germany) was used for amplification as per the manufacturer's manual. Marker ge 16S rRNA was amplified using forward and reverse primer. Forward primer 27F sequence used was (5'-AGA GTT TGA TCC TGG CTC AG-3') and reverse primer 1492R sequence was (5'-GGT TAC CTT GTT ACG ACT T-3′) according to Bergmann et al. (2010). The control excluded DNA, according to Sambrook and Russell, (2001). Response combinations were exposed to the accompanying temperature cycling profiles rehashed for 32 cycles: Initial denaturation 94°C for 5 minutes, denaturation at 94°C for one minute, groundwork strengthening at 55°C for 2 minutes, expansion at 72°C for 2 minutes and the last augmentation at 72°C for 10 minutes. Purification of PCR products was done using QIAquick PCR purification Kit protocol (Qiagen, Germany) according to the manufacturer's instructions. Polymerase chain reaction purified amplicons were run on 1% (w/v) molecular grade agarose gel mixed with 1μg/ml ethidium bromide in 1X TBE cradle according to ref 17 .
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