Genomic DNA of MCF-7 wt, Control KO, and Bag-1 KO cells (1X106) was extracted using Quick DNA/RNA Kit (Zymo Research). Primers that have the binding sites of sgRNAs were designed for exon 2, exon 3, and exon 6 (S1A Table ). To detect the off-target effects, 3 different primers were also designed according to the most possible off-target sites. Primers were designed using websites CRISPOR (http://crispor.tefor.net/crispor.py ) and Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi ), and were synthesized by Thermo Scientific, USA. All primers were listed in S1B Table . Specific regions of Bag-1 from MCF-7 cells were amplified by using Taq DNA Polymerase (Thermo Scientific, USA). PCR amplicons were run on %2 agarose gel. PCR products were Sanger sequenced with Applied Biosystems 3500XL Genetic Analyzer for Human Identification (Thermo Scientific, USA).
Applied biosystems 3500xl genetic analyzer for human identification
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 3500XL Genetic Analyzer is a capillary electrophoresis system designed for human identification applications. The instrument utilizes advanced optical and fluidic technologies to perform high-resolution DNA fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Quick dna rna kit, by Zymo Research (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Maxwell rsc instrument, by Promega (1 mentions)
Plexor hy kit, by Promega (1 mentions)
Applied biosystems veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
2 protocols using applied biosystems 3500xl genetic analyzer for human identification
1
Analyzing Genomic Alterations in Breast Cancer Cells
2
Standardized DNA Extraction and Amplification
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All 300 samples were extracted using the DNA IQ™ Casework, and Extraction Kit (Promega Corporation) loaded on a Maxwell® RSC Instrument (Promega) with 50 µl elution buffer [36, 37] . DNA was quantified once with the Plexor® HY Kit (Promega) on an Applied Biosystems™ 7500 Real Time PCR System (Thermo Fisher Scientific) according to the manufacturers' protocols [38, 39] . Single STR amplification was done with the PowerPlex® ESX 17 Fast System Kit (Promega) on a 96-well Applied Biosystems Veriti™ Thermal Cycler (Thermo Fisher Scientific) according to the manufacturers' instructions [40, 41] . The kit amplifies the following loci, including the extended European Standard Set D18S51, D21S11, TH01, D3S1358, D16S539, D2S1338, D1S1656, D10S1248, FGA, D8S1179, vWA, D22S1045, SE33, D19S433, D12S391, D2S441 and Amelogenin [42] (link)[43] (link)[44] . PCR products were analyzed on the capillary fragment analyzer Applied Biosystems™ 3500xL Genetic Analyzer for Human Identification (Thermo Fisher Scientific, [45] ).
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