Five microliter of 100nM circular single-stranded M13 DNA (250 μg/ml, New England Biolabs), 2.5 μl of 10 × Cut Smart buffer (New England Biolabs), 0.5 μl of 100 μM BtsCI restriction-site complementary-oligonucleotide and 16 μl of deionized water were mixed and annealed from 95°C to 50°C in a T100™ Thermal Cycler (Bio-Rad, USA). One microliter of the BtsCI enzyme (20 000 units/ml, New England Biolabs) was added to the mixture and incubated at 50°C for 15 min. The mixture was brought up to 95°C for 1 min to heat deactivate the enzyme followed by cooling down to 4°C.
Btsci enzyme
Manufactured by New England Biolabs
BtsCI is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-GCAGTG-3'/3'-CGTCAC-5'. It is a useful tool for molecular biology applications such as DNA cleavage and fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotide, by Integrated DNA Technologies (2 mentions)
Gelred nucleic acid stain, by Biotium (2 mentions)
Molecular biology grade agarose, by Thermo Fisher Scientific (2 mentions)
M13 circular dna, by New England Biolabs (2 mentions)
Rnase h, by New England Biolabs (2 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
4 protocols using btsci enzyme
1
M13 DNA Restriction Digestion
2
Molecular Detection of Antimicrobial Resistance Genes
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PCR assays were performed on all isolates for the detection of carbapenemase genes (blaNDM, blaVIM, blaIMP, blaSPM, blaGIM, blaSIM,blaKPC,blaOXA-48) [8 (link), 11 (link)–13 (link)], other β-lactamase genes (blaCTX-M, blaTEM, blaSHV, blaOXA-1) [14 (link), 15 (link)] and PMQR genes (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib-cr, qepA, oqxA, oqxB) [3 (link), 16 (link)]. The qepA and aac(6′)-Ib-cr genes were analyzed by a multiplex PCR with a buffer suitable for GC rich sequences as the GC content of qepA gene is high (70%). The aac(6′)-Ib-cr was differentiated from its wild-type allele by digestion with BtsCI enzyme (New England Biolabs, Massachusetts) [16 (link)]. Primers used do not discard the presence of the non-ESBL variants of blaTEM and blaSHV. As the study focuses on blaNDM-1 and PMQRs, the PCR products of blaTEM and blaSHV genes were not further sequenced and this remains a shortcoming of the study.
3
M13mp18 DNA Nanoswitches Construction
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Oligonucleotides were purchased from Integrated DNA Technologies (IDT) with standard desalting. M13 circular DNA, RNase H, RNase T (Exo T), RNase If, and BtsCI enzymes were purchased from New England Biolabs (NEB). GelRed nucleic acid stain was purchased from Biotium. Molecular biology grade agarose was purchased from Fisher BioReagents. We have used the viral genome M13mp18 (7,249 nt) for this and previous constructions of our nanoswitches due to its commercial availability and frequent use in DNA origami.
4
M13mp18 DNA Nanoswitches Construction
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