The main research materials of this study consists of High Pure Viral Nucleic Acid Kit (cat. no: 11-858-874-001) from Roche, SuperScript™ III One-Step RT-PCR with Platinum Taq (cat. no: 12574-026) from Invitrogen, UltraPure™ agarose gel from (cat. no:16500-100) from Invitrogen, 1x Buffer Tris base-Boric acid-EDTA, PBS, RedSafe nucleic acid dye from Intron, aquabidest, distilled water, Blue Loading dye (cat. no: 10816-015), 1 kb DNA Ladder (cat. no: 29,278,801) from Promega, and the specific oligonucleotide primers (Table-2 ).
Ultrapure agarose gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
UltraPure™ agarose gel is a high-quality laboratory product used for DNA and RNA separation and analysis. It is designed to provide consistent and reliable performance in gel electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc xr imager, by Bio-Rad (3 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
Mastercycler gradient thermocycler, by Eppendorf (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Uv transilluminator, by Bio-Rad (1 mentions)
Whatman, by Cytiva (1 mentions)
Primestar gxl polymerase, by Takara Bio (1 mentions)
E z n a cycle pure kit, by Omega Bio-Tek (1 mentions)
Qiasymphony, by Qiagen (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop one microvolume spectrophotometer, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Platinum pcr supermix high fidelity, by Thermo Fisher Scientific (1 mentions)
Noti restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Low melting temperature agarose, by Thermo Fisher Scientific (1 mentions)
Chef dr 3 apparatus, by Bio-Rad (1 mentions)
16 protocols using ultrapure agarose gel
1
Viral RNA Extraction and RT-PCR Protocol
2
Genomic DNA Extraction and Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
Genomic DNA was extracted from peripheral blood
leucocytes by the salting out method. The fragments
harboring the selected SNPs were amplified by PCR with
the use of specific oligonucleotide primers designed by
Perl primer version1.1.20 (Table 1 ). PCR amplification
was performed in a 25 μl reaction mixture containing 1.5
mM MgCl2, 0.2 mM dNTP, 1X PCR buffer, 0.06 U/μl Taq
DNA polymerase enzyme (all from Cinagen, Iran), 0.4
pmol of each primer (Fazapajoh, Iran) and 2 μl of the DNA
template. The PCR conditions for all except the promoter
region SNP (the-29G>A polymorphism) were an initial
DNA denaturation at 95˚C for 5 minutes, followed by
30 cycles of DNA denaturation at 95˚C for 45 seconds,
annealing at melting temperature (TM) for 45 seconds
(Table 1 ) and extension at 72˚C for 45 seconds followed
by a final extension at 72˚C for 10 minutes. The PCR
cycling conditions for the promoter SNP were 4 minutes
of initial DNA denaturation at 94˚C followed by 30 cycles
that consisted of 45 seconds of denaturation at 94˚C, 45
seconds of annealing at 64˚C and 45 seconds of extension
at 72˚C followed by 8 minutes for final extension. All
PCR reactions were performed in a master cycler gradient
thermocycler (Eppendorf, Germany). All PCR products
were run on a 1.7% ultra-pure agarose gel (Invitrogen,
USA), stained by ethidium bromide (Invitrogen, USA)
and visualized under the UV light.
+ Expand
Genomic DNA was extracted from peripheral blood
leucocytes by the salting out method. The fragments
harboring the selected SNPs were amplified by PCR with
the use of specific oligonucleotide primers designed by
Perl primer version1.1.20 (
was performed in a 25 μl reaction mixture containing 1.5
mM MgCl2, 0.2 mM dNTP, 1X PCR buffer, 0.06 U/μl Taq
DNA polymerase enzyme (all from Cinagen, Iran), 0.4
pmol of each primer (Fazapajoh, Iran) and 2 μl of the DNA
template. The PCR conditions for all except the promoter
region SNP (the-29G>A polymorphism) were an initial
DNA denaturation at 95˚C for 5 minutes, followed by
30 cycles of DNA denaturation at 95˚C for 45 seconds,
annealing at melting temperature (TM) for 45 seconds
(
by a final extension at 72˚C for 10 minutes. The PCR
cycling conditions for the promoter SNP were 4 minutes
of initial DNA denaturation at 94˚C followed by 30 cycles
that consisted of 45 seconds of denaturation at 94˚C, 45
seconds of annealing at 64˚C and 45 seconds of extension
at 72˚C followed by 8 minutes for final extension. All
PCR reactions were performed in a master cycler gradient
thermocycler (Eppendorf, Germany). All PCR products
were run on a 1.7% ultra-pure agarose gel (Invitrogen,
USA), stained by ethidium bromide (Invitrogen, USA)
and visualized under the UV light.
3
Dried Blood Spot DNA Extraction and PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three drops, each containing 50 μL of EDTA blood were spotted on a pre-made filter paper (Whatman 3MM, Maidstone, UK) and allowed to dry at room temperature. The filter papers were placed in zip-locked, plastic bags containing silica gel to preserve DNA integrity and stored at −20 °C and then transported to Republic of Korea for molecular analysis. DNA was extracted from half-segment of the filter spot using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer, and eluted in a final volume of 100 μL. The extracted DNA was analysed using P. falciparum species-specific PCR targeting mitochondrial genome 18S as previously described by Kho et al. [21 (link)]. The parasite DNA amplification was performed using conventional PCR 96 well Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA). The PCR products were analysed in 1.5 % ethidium bromide stained UltraPure™ agarose gel (Invitrogen) with a Gene ruler™ 100 bp DNA ladder (TaKara Bio Inc, Shiga, Japan). The gels were visualized under UV transilluminator from BIO-RAD.
4
PCR Amplification of Target Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was performed using Lac-Forward (5′-CTTCAAATCCGACCCGTAGA-3′) and TK-Reverse (5′-GTAAGTCATCGGCTCGGGTA-3′) primers. PrimeSTAR GXL polymerase (Takara) was used per manufacturer's instructions for 50 μl reactions using 120 ng template. Internal control primers (Figure 3 ) were: forward 5′-CCCAACCTACACTAACCTTAACC and reverse 5′-CCACACCAACCTCCTCATAAT. Cycling conditions were as follows: denaturation, 98°C, 10 s; annealing, 57°C, 15 s; extension, 68°C, 1 min for 30 cycles. PCR products were purified with an EZNA Cycle Pure kit (Omega Bio-Tek) and 20 μl of each purified product was electrophoresed on a 1% ultra-pure agarose gel (Invitrogen) to verify the sizes of the recombination products.
5
Automated Extraction and Characterization of cfDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was isolated from thawed mammalian cell culture medium supernatant through automated nucleic acid extraction using the QIAsymphony® (Qiagen). Fifty microliters elutes from the same culture medium were pooled and subsequently quantified using the Qubit™ dsDNA HS kit (Invitrogen, Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Invitrogen, Thermo Fisher Scientific). Isolates were stored at −20°C. Mock cfDNA samples derived from cell culture were separated on a 1.5% UltraPure™ agarose gel (Invitrogen, Thermo Fisher Scientific) with 0.05% (v/v) EtBr and visualized with a Gel Doc XR+ imager (Bio-Rad Laboratories, Inc.) to confirm presence of expected nucleosomal fragment sizes.
For the tumor fraction simulation assay, mock cfDNA from VU1131 was diluted with mock cfDNA obtained from a TP53 wild-type cell line (WM9). Tissue biopsies were subjected to macrodissection before DNA isolation, ensuring that neoplastic cellularity was at least 20%. Subsequent DNA isolation was done using the Purelink™ Genomic DNA Mini Kit (Cat. No. K182001; Invitrogen, Thermo Fisher Scientific). Nucleic acid concentration and purity were assessed using a NanoDrop One microvolume Spectrophotometer (Thermo Fisher Scientific).
For the tumor fraction simulation assay, mock cfDNA from VU1131 was diluted with mock cfDNA obtained from a TP53 wild-type cell line (WM9). Tissue biopsies were subjected to macrodissection before DNA isolation, ensuring that neoplastic cellularity was at least 20%. Subsequent DNA isolation was done using the Purelink™ Genomic DNA Mini Kit (Cat. No. K182001; Invitrogen, Thermo Fisher Scientific). Nucleic acid concentration and purity were assessed using a NanoDrop One microvolume Spectrophotometer (Thermo Fisher Scientific).
6
Inverse PCR Identifies Acinetobacter Transposon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Inverse PCR was used to identify the transposon insertion site of A. baumannii strain ACJ7, as previously described [17] (link). Briefly, 2 µg of ACJ7 genomic DNA was purified using DNeasy® Blood and Tissue Kit following the manufacturer's recommendations (Qiagen, Valencia, CA), and then digested by restriction enzyme AfeI (10 U; New England Biolabs) at 37°C for 1 hr. Restriction fragments were circularized by ligation using 1.5 U of T4 DNA ligase (Invitrogen) at 16°C for 16 h and were subjected to inverse PCR using Platinum® PCR Supermix High Fidelity (Invitrogen) and transposon-specific primers (forward: 5′-ACCTACAACAAAGCTCTCATCAACC-3′ ; and reverse: 5′-CTACCCTGTGGAACACCTACATCT-3′ ) supplied in the EZ-Tn5™ ori/KAN-2> Tnp transposome kit (Epicentre Biotechnologies). PCR products were electrophoresed in a 1% UltraPure™ Agarose gel (Invitrogen), purified using a QIAquick® Gel Extraction Kit (Qiagen), ligated into pCR®II-TOPO® and transformed into E. coli One Shot® INVαF' cells, following the manufacturer's recommendations for Dual Promoter TA Cloning (Invitrogen). Following propagation, plasmid DNA was purified using QIAprep® Spin Miniprep Kits (Qiagen) and sequenced at the University of Nebraska Medical Center Sequencing Core Facility using vector specific primers (forward: 5′ -GTAAAACGACGGCCAG-3′ ; reverse: 5′ -CAGGAAACAGCTATGAC-3′ ).
7
Pulsed-field gel electrophoresis typing of Proteus mirabilis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the phenotypic and genotypic results, thirty-three isolates were selected from the 110 P. mirabilis isolates for PFGE typing. Briefly, isolates were diluted in a buffer (75 mM NaCl and 25 mM EDTA) and mixed with 2% low-melting-temperature agarose (Invitrogen, USA). Then, 100 µL of the mixture was loaded in plug molds and allowed to set at 4°C. Plugs were incubated with lysis buffer containing 0.5 M EDTA, 1% N-lauryl sarcosine, and proteinase K at 50°C. Then, plugs were washed, digested with NotI restriction enzyme (Fermentas, Lithuania), and loaded on 1% ultrapure agarose gel (Invitrogen, USA) wells. Electrophoresis was carried out with CHEF-DR III apparatus (Bio-Rad, USA), and DNA fragments were separated by electrophoresis at 6 V/cm for 24 hrs with 2 blocks, the first block switch time was 1 to 30 s for 8 hrs and the second block was 30 to 70 s for 16 hrs. Salmonella enterica serovar Branderup strain H9812 (ATCC BAA-664) was used as size marker. The band patterns were analyzed with Gel compare II software (Applied math, Belgium). Furthermore, clonal relationship between the tested isolates was evaluated by using the unweighted pair group method with arithmetic mean (UPGMA). Relatedness of isolates was defined on the basis of PFGE profiles with ≥80% similarity.21 (link)
8
Lyophilization and Genomic DNA Extraction
9
Efficient DNA Size Estimation
10
PCR Amplification of TP53 Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR amplification of TP53 loci containing the p.R273 codon, or mutants thereof, was done using primers KD046 and KD047, which enclose the first 73 base pairs of exon 8. Using Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs), a final template concentration of 0.2 ng/μL and an annealing temperature of 68°C for an indicated amount of cycles, 73 bp amplicons were synthesized with codons of interest in the middle. Reaction products were visualized on a 1.5% UltraPure agarose gel (Invitrogen, Thermo Fisher Scientific) with 0.05% (v/v) EtBr and visualized with a Gel Doc XR+ imager (Bio-Rad Laboratories, Inc.).
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!