mRNA cleavage fragments were evaluated from 1 μg RNA isolated from either vehicle or EGFR1-Cent-ABD-siRNA_1-treated tumor tissue with a 5′ RACE assay using the Invitrogen GeneRacer Kit with SuperScript III RT and TOPO TA Cloning Kit for Sequencing (L150201), per the manufacturer’s protocol. PCR amplification was done with the Gene Racer 5′ nested primer and gene-specific inner primer (5′-ACCCCCTCCACAAATTGCTGCTGTGT-3′). The PCR product from tumor tissue isolated from mice treated with vehicle or 83v2-ABDcon12-siRNA (5 μL/sample) was run on an agarose gel. Sample was added to 1 μL of 10× Blue Juice (Invitrogen) and loaded on a 1% agarose gel (BioRad) in 1× Tris-borate-EDTA (TBE) (Invitrogen). Track IT 1 kB Plus DNA ladder (Invitrogen) was also run, along with 5 μL of the BioRad ladder. Gel was run at 125 V for 45 min, imaged using a BioRad Gel Doc XR, and analyzed with BioRad Image Lab software. Relevant bands were cut out, and mRNA was extracted from the gel using the Wizard PCR and Gel Cleanup Kit (Promega). Fragments were eluted in 50 μL nuclease-free water and cloned in Invitrogen’s TOPO TA cloning vector, using TOPO TA Cloning Kit for Sequencing (Invitrogen).
Wizard pcr and gel cleanup kit
Manufactured by Promega
Sourced in United States
The Wizard PCR and Gel Cleanup Kit is a product designed to purify DNA from PCR amplifications and agarose gel slices. It utilizes a silica-based membrane technology to bind and concentrate DNA, allowing for the removal of unincorporated primers, nucleotides, enzymes, and other contaminants.
Automatically generated - may contain errors
Lab products found in correlation
Topo ta cloning kit for sequencing, by Thermo Fisher Scientific (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Generacer kit with superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Blue juice, by Thermo Fisher Scientific (1 mentions)
Track it 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Genomiphi v2 kit, by GE Healthcare (1 mentions)
4 protocols using wizard pcr and gel cleanup kit
1
EGFR1-Cent-ABD-siRNA Treatment and mRNA Cleavage Analysis
2
Identification of Fusarium Species from Banana
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DNA was extracted from the cultures of Fusarium isolated from banana tissue using the process previously described. To ensure that (i) the DNA was of sufficient quality for PCR, and (ii) the cultures were Fusarium species, amplification of a region in the EF1-α was conducted as previously described. Isolates that had an EF1-α amplicon of the expected size were further assessed for SIX genes using the optimised universal primers. Amplification of SIX1–SIX14 was performed using the optimised conditions described above and in Table 4 . Amplicons were size separated and visualised as previously described. The EF1-α and SIX gene amplicons of the expected sized were purified using the Wizard PCR and Gel Clean Up kit (Promega Corporation, WI, USA) and sequenced by Macrogen (Macrogen Inc., Seoul, Korea) using the primers used for amplification of the target gene. The resulting EF1-α and SIX gene sequences were deposited to GenBank (accessions MW076542-MW076821).
3
Viral DNA Amplification and Sequencing
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Independent from the procedure used for viral recovery, the amounts of viral DNA isolated from the different benthic deep-sea sites with the exception of the Black Sea were not sufficient to be directly sequenced. Therefore, for a proper comparison all samples were amplified by Multiple Displacement Amplification (MDA) to increase the concentration of viral DNA suitable for metagenomic analysis51 (link). Samples were processed with GE Healthcare GenomiPhi V2 kit (according to the manufacture instructions). After MDA, viral DNA was purified (using Wizard PCR and Gel Clean-up kit, Promega) and then sequenced onto a 454 Titanium FLX platform. To assess the potential biases induced by MDA, we compared MDA treated and un-treated viromes generated from sediment samples collected in the same deep-sea site of the Mediterranean Sea. To do so, DNA of viruses extracted by the PC procedure applied on 500 g of sediment was split into two aliquots, one of which amplified by MDA and the other was left untreated. Both DNA pools were then sequenced as reported above.
4
Full-length dCK gene sequencing
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The full open reading frame of dCK was PCR-amplified from cDNA in two segments (primers 1 + 4 and 3 + 8, Supplementary Table 1 ) as described above. PCR products were purified using the Wizard PCR and gel cleanup kit (Promega, Fitchburg, WI, USA) and sequenced at Hylabs laboratories (Rehovot, Israel) with an ABI 3730xl DNA Analyzer using BigDye Terminator 1.1 Cycle Sequencing Kit (ABI).
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