Total genomic DNA was extracted from the NRE isolates using an Invitrogen Genomic DNA kit. Amplify of nifH gene were achieved using Zehr-nifHf (5′-TGYGAYCCNAARGCNGA-3′), and Zehr-nifHr (5′-ADNGCCATCATCATCATYTCNCC-3′), primers (Rashid et al., 2012 (link)). The amplification was carried out in 50 µl reaction mixtures, each comprising 1 µl of forward and reverse primer (10 M concentration), 25 µl of Taq Green Master Mix (Invitrogen, India), and 22 µl of nuclease-free water at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, following 72°C for 45 s, and final extension at 72°C for 10 min. The amplicons were detected on 1% agarose gels containing TBE buffer. For further confirmation, acetylene reductase assay was performed to evaluate the ability of the isolates to fix atmospheric nitrogen (Kaushal and Kaushal, 2015 ).
Genomic dna kit
Manufactured by Thermo Fisher Scientific
The Genomic DNA kit is a laboratory product designed to isolate and purify high-quality genomic DNA from a variety of sample types. It employs a simple and efficient process to extract DNA, which can then be used for various downstream applications such as PCR, sequencing, and genomic analysis.
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4 protocols using genomic dna kit
1
Amplification and Evaluation of nifH Gene
2
CRISPR-Cas9 Editing of HEB in hESCs
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The pD1321-GFP expression vector containing cassettes for GFP, Cas9 endonuclease, and a CRISPR chimeric cDNA including the guide RNA moiety designed to target exon 9 of HEB (CCA GTA TGT TCG CTA GCA CTT TC), was custom synthesized (DNA2.0). hESCs were dissociated into single cells using Accutase (Sigma), and transfected with the CRISPR/Cas9 vector using the Nucleofector 2B device, program A013 (Amaxa). GFP+ cells were sorted 48 hr after transfection using a FACSAria II cell sorter (BD Biosciences) at the Sunnybrook Research Institute (SRI) Center for Flow Cytometry & Microscopy. Single cells were cultured on Matrigel-coated plates in TeSR-E8 medium, which was supplemented with Rho kinase (ROCK) inhibitor Y-27632 (Tocris) for the first 24 hr. Individual colonies were picked and expanded under the feeder-free/TeSR-E8 conditions. Aliquots of the cells were collected for purification of genomic DNA using the Genomic DNA kit (Invitrogen). Mutations were validated by sequencing the PCR amplification products of the regions flanking the targeting sites.
3
CRISPR-mediated RAG2 Knockout in hESCs
4
Genomic DNA Extraction and PCR Amplification
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Genomic DNA was extracted from peripheral blood leukocytes using Genomic DNA kit (Invitrogen). All exon regions, including flanking introns and promoters of GCK (NM_000162.5) and HNF1A (NM_000545.6) genes were amplified by polymerase chain reaction (PCR), by using the previously reported primers [11 (link)].
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