Five mg of lyophilised Amolops wuyiensis skin secretion was dissolved in 1 mL of Lysis/Binding buffer. A Dynabeads® mRNA DIRECT™ Kit (Dynal Biotech, Merseyside, UK) was used for mRNA isolation. The cDNA library construction was performed using Clontech SMARTer® RACE 5′/3′ Kit (Takara Bio, USA, Inc., San Jose, CA, USA). A nested universal primer (NUP) and a degenerate sense primer (S1: 5′-GAWYYAYYHRAGCCYAAADATG-3′; W = A + T, Y = C + T, H = A + C + T, R = A + G, D = A + G + T) were used. The cDNA ends were rapidly amplified by a PCR thermal cycling system with repeated denaturation, annealing, and extension. The products were analysed by gel electrophoresis and then purified using a Hi-Bind DNA mini-column (Omega Bio-Tek, Norcross, GA, USA). The process of DNA ligation was performed using a pGEM-T Easy Vector System (Promega Corporation, Madison, WI, USA). After that, the recombinant plasmid DNA was cloned in JM109 high-efficiency competent cells (Promega, Madison, WI, USA). A Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for sequencing reaction and the products were analysed using an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).
Hi bind dna mini column
Manufactured by Omega Bio-Tek
Sourced in United States
The Hi-Bind DNA mini-column is a laboratory equipment used for the isolation and purification of DNA samples. Its core function is to selectively bind DNA molecules while allowing other cellular components to be removed through a series of washing steps. The mini-column design provides an efficient and convenient method for DNA extraction and purification.
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Lab products found in correlation
Clontech smarter race 5 3 kit, by Takara Bio (1 mentions)
Jm109 high efficiency competent cells, by Promega (1 mentions)
Abi 3730 automated sequencer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Smart race kit, by Takara Bio (1 mentions)
Abi 3100 automated sequencer, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector kit, by Promega (1 mentions)
3 protocols using hi bind dna mini column
1
Amolops wuyiensis Skin Secretion cDNA Library Construction
2
Oyster Hemolymph DNA Extraction
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One to two ml of hemolymph was drawn from each oyster's adductor muscle with a 1 ml syringe fitted with a 27 gauge needle. The hemolymph was diluted in 3 vol of 1X PBS and centrifuged at 4000 × g for 10 min to pellet the hemocytes. The cell pellet was washed once in 1X PBS and then resuspended in 400 µl of hypotonic buffer (50 mM Tris–HCl pH 8.0, 5 mM EDTA, 50 g/ml RNAse, 2 % Triton X-100) at 4 °C for 24 h (19). Cell nuclei from the post-hypotonic treatment were pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was loaded directly to the HiBind DNA Mini Column (Omega Bio-tek). The DNA binding column was washed according to the manufacturer's instructions for the EZNA Tissue DNA Kit, and DNA was eluted in 50 µl of TE buffer. Around 10–50 ng of DNA isolated from the supernatant was used in the PCR for detection of OsHV-1. Total DNA from the pellet of the hypotonically-treated cell nuclei was isolated according to the manufacturer's instructions for the EZNA Tissue DNA Kit, and the DNA was also eluted in 50 µl of TE buffer. Approximately 100–500 ng of DNA isolated from the cell pellet was used in the PCR.
3
Palustrin-2LTb Peptide Sequencing and Identification
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‘Shotgun cloning’ and sequencing were conducted to determine the nucleotide sequence of palustrin-2LTb. mRNA was separated from Hylarana latouchii skin secretion by use of a Dynabeads mRNA Direct Kit (Dynal Biotech, UK). Poly-A mRNA was attracted to the oligo (dT) beads. After elution, all the mRNAs were acquired. The first strand cDNA was obtained by reverse transcription, and then a cDNA library was constructed. A SMART-RACE kit (Clontech, UK) was employed to obtain the DNA sequence of prepro-peptide nucleic acids using a NUP primer (supplied with the kit) and a degenerate sense primer (S1:50-GAWYYAYYHRAGCCYAAADATGTTCA-30). After that, quick amplification of cDNA ends polymerase chain reaction (RACE-PCR) was utilised to amplify the cDNA; denaturation, annealing and extension were repeated for thermal cycling. The products were separated through gel electrophoresis and purified by a Hi-Bind DNA mini-column (Omega BIO-TEK, USA). Purified PCR was ligated to the vector using a pGEM-T easy vector kit (Promega, USA) and sequenced by an ABI 3100 automated sequencer (Applied Biosystems, USA). The translated open reading frame was compared to similar sequences using the National Centre for Biotechnology Information (NCBI) BLAST programme. Sequences with high identities were then aligned with the novel peptide.
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