The deduced nucleotide sequence of avimer molecule was synthesized and cloned into pGH cloning vector by GENEray Co. (Shanghai, China). The pET-26b(+) expression vector with kanamycin resistance gene was used for cloning and expression purpose. The DNA fragment of C426 avimer was excised from pGH cloning vector using NcoI and HindIII restriction enzymes and was recovered by gel purification kit (Bioneer, South Korea; cat. no. k-3035) according to the manufacturer’s instructions. Then, it was sub-cloned into linear pET-26b(+) cloning vector by T4 DNA ligase (Vivantis, USA). The E. coli strain DH5α competent cells were prepared by TSS reagent and recombinant plasmid was transformed using heat shock method. For screening, the transformed cells were plated on LB-agar medium containing kanamycin antibiotic (50 μg/ml). Recombinant plasmid was purified using the plasmid extraction kit (Bioneer, South Korea; cat. no. k-3112). The size of insert was validated by restriction digestion analysis with NcoI and HindIII and electrophoresis on 1.5% agarose gel. The precision of the ORF region was verified by sequencing analysis (GENEray, China).
T4 dna ligase
Manufactured by Vivantis Technologies
Sourced in Malaysia
T4 DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded DNA. It is commonly used in molecular biology and genetic engineering applications to join DNA fragments together.
Automatically generated - may contain errors
Lab products found in correlation
Gel purification kit, by Bioneer (1 mentions)
Plasmid extraction kit, by Bioneer (1 mentions)
Endoglycosidase h endo h, by New England Biolabs (1 mentions)
Sucrose, by Merck Group (1 mentions)
Bamhi enzyme, by Thermo Fisher Scientific (1 mentions)
Ptg19 t vector, by Vivantis Technologies (1 mentions)
4 protocols using t4 dna ligase
1
Cloning and Expression of Avimer Protein
2
Characterization of Glycosidic Enzymes
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DNA restriction enzymes and T4 DNA Ligase were purchased from Vivantis Technologies Co. (Malaysia). Taq DNA polymerase, DNA Ladders and Protein markers were from Fermentase Thermo Fermentase (USA). Endoglycosidase H (Endo H) was purchased from New England Biolabs (Beverly, MA, USA).
Native invertase was purchased from Sigma-Aldrich (USA) for using as positive control enzyme in kinetic experiments. Oligonucleotides were synthesized at TAD Copenhagen A/S (Denmark). Sucrose was from Merck (Darmstadt, Germany) and other chemicals were obtained from Merck (Darmstadt, Germany) or Sigma Chemical Co. (St Louis, MO, USA).
Native invertase was purchased from Sigma-Aldrich (USA) for using as positive control enzyme in kinetic experiments. Oligonucleotides were synthesized at TAD Copenhagen A/S (Denmark). Sucrose was from Merck (Darmstadt, Germany) and other chemicals were obtained from Merck (Darmstadt, Germany) or Sigma Chemical Co. (St Louis, MO, USA).
3
Cloning and Sequencing of RT-PCR Amplicons
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Amplified fragments resulting from the reverse transcription polymerase chain reaction (RT-PCR) were ligated into the pTG19-T vector (Vivantis, Malaysia) using 1 Unit of T4 DNA ligase (Vivantis, Malaysia) according to the manufacturer’s protocol and incubated at 4°C overnight. Ligation mixes were transformed into prepared chemically competent Escherichia coli strain DH5α cells by heat shock method (Chung et al., 1989 (link)). The transformed cells were selected on LB plates containing ampicillin (100 mg/ml), IPTG (100 μl of 0.1 M per plate), and X-Gal (20 μl of 50 mg/ml). Plasmids were extracted by Accuprep nanopluse plasmid mini extraction kit (Bioneer, South Korea) according to manufacturer’s instruction. The recombinant plasmids were verified by PCR colony and each colony carrying the cloned cDNA was subjected to single colony isolation. Also, the purified plasmids were digested by BamHI enzyme (Thermo Fisher Scientific, USA) to release the DNA inserted and loaded on 1% agarose gel. Next, the independent clones of each viral isolate were subjected to nucleotide sequencing with M13F/R primers. The sequencing was done by Bioneer Inc. (Seoul, South Korea).
4
DNA Fragment Isolation and Sequencing
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Digested fragments were isolated and extracted by
GEL DNA recovery kit (Vivantis, Malaysia), and then
ligated with T4 DNA ligase (Vivantis, Malaysia) for 4
hours at 16ºC. Eventually, ligated DNA was reamplified
and directly sequenced along with undigested wild-type
fragment using the same primer pairs used in PCR-
restriction-fragment length polymorphism (PCR-RFLP)
(Table 1 ).
GEL DNA recovery kit (Vivantis, Malaysia), and then
ligated with T4 DNA ligase (Vivantis, Malaysia) for 4
hours at 16ºC. Eventually, ligated DNA was reamplified
and directly sequenced along with undigested wild-type
fragment using the same primer pairs used in PCR-
restriction-fragment length polymorphism (PCR-RFLP)
(
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