All PCR products were digested with the respective enzymes in 30 µl reaction volumes as follows: 10 µl of unpurified PCR product, 2 µl of Green Buffer (to directly load digested PCR products on agarose gels), 1 µl of either FastDigest Eco321 (isoschizomer or EcoRV), FastDigest NdeI or FastDigest PvuII (all Thermo Scientific), and 17 µl deionized water. Incubation was conducted at 37 °C for 5 min for Eco321 and PvuII, and for 60 min for NdeI. Digested PCR products were visualized on 2% TBE agarose gels and compared to a 100 to 1000 bp ladder to determine the size of the fragments. Species were identified by comparing the resulting patterns to the expected fragment lengths (Table 2 ).
Fastdigest ndei
Manufactured by Thermo Fisher Scientific
FastDigest NdeI is a restriction enzyme that recognizes and cleaves the DNA sequence 5'-CATATG-3'. It is a fast-acting enzyme designed for rapid DNA digestion. The core function of FastDigest NdeI is to facilitate the site-specific cleavage of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Q5 reaction buffer, by New England Biolabs (1 mentions)
Fastdigest xhoi, by Thermo Fisher Scientific (1 mentions)
Plasmid miniprep kit, by Evrogen (1 mentions)
Cleanup standard kit, by Evrogen (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
2 protocols using fastdigest ndei
1
PCR Fragment Analysis by Restriction Digestion
2
Bacterial Expression of NS1 Protein
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-3′), containing an Xho1 restriction site. PCR was performed using a C1000 Thermal Cycler (Bio-Rad), Q5 High-Fidelity DNA Polymerase (NEB, M0491S), and 5x Q5 Reaction Buffer (NEB, B9027S) according to manufacturer protocols (53°C annealing temp.). Following electrophoretic separation in 1% agarose (in TBE), DNA bands were excised with subsequent purification of PCR products using a Cleanup Standard Kit (Evrogen, BC022). Restriction enzymes, namely 'FastDigest XhoI' and 'FastDigest NdeI' (Thermo Fisher Scientific, FD0695 and FD0585, respectively), were used to prepare sticky ends in insert and vector. The pET-22b+ vector (Novagen, USA) was used to make our construct (pET-NS1). Next, ligation was performed using T4 DNA Ligase (NEB, M0202L), and DH5a bacterial cells were electroporated with ligation mixes. Subsequent growth on ampicillin plates (100 ug/ml) enabled clone selection. After selection, pET-NS1 was purified using a Plasmid Miniprep Kit (Evrogen, BC021). Commercial plasmid sequencing was performed using T7 primers at Evrogen (Moscow, http://evrogen.com). Amino acid sequence corresponding to the coding region of the constructed plasmid is shown in Table 1. After sequence verification, the BL21(DE3) E. coli strain was transfected with pET-NS1 plasmid using standard protocols.
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