Chroma spin depc h2o columns

Manufactured by Takara Bio

Sourced in United States

The Chroma Spin DEPC-H2O columns are a laboratory product designed for the removal of RNase contamination from water samples. These columns utilize a proprietary resin to effectively eliminate DEPC (diethyl pyrocarbonate), a common RNase inhibitor, from the water, ensuring a high-quality, RNase-free water supply for various molecular biology applications.

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Lab products found in correlation

T7 mmessage machine kit, by Thermo Fisher Scientific (2 mentions) Rneasy rna purification kit, by Qiagen (2 mentions) T4 dna ligase, by New England Biolabs (2 mentions) Full length 5 and 3 dna strands, by Integrated DNA Technologies (2 mentions) T7 scribe transcription kit, by CellScript (2 mentions) Megashortscript t7, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Chroma spin columns (5 citations) DEPC H2O (3 citations)

4 protocols using chroma spin depc h2o columns

Site-Directed Mutagenesis of rat nAChR α7

Cited 1 time

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The QuikChange protocol (Stratagene) was used for site-directed mutagenesis of the rat nAChR α7 subunit (pAMV vector). NotI was used to linearize the circular plasmid. The DNA was purified (Qiagen) and in vitro transcription of mRNA from the linearized DNA templates was performed using the T7 mMessage Machine kit (Ambion). The resulting mRNA was purified and isolated using Qiagen’s RNeasy RNA purification kit. The same linearization and mRNA synthesis protocols were used for the human Ric3 (pAMV) accessory protein.
For non-canonical amino acid incorporation, the amber (UAG) stop codon was used for all α7 subunit incorporation. The 74-nucleotide THG73 tRNA and 76-nucleotide THG73 tRNA were in vitro transcribed using the MEGAshortscript T7 (Ambion) kit and isolated using Chroma Spin DEPC-H₂O columns (Clontech). Synthesized non-canonical amino acids coupled to the dinucleotide dCA were enzymatically ligated to the 74-nucleotide tRNA as previously described (Nowak, et al., 1998 (link); Xiu, et al., 2009 (link)).
ND96 media was used for all experimental running/wash buffers (96 mM NaCl, 1.8mM CaCl₂, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES at pH 7.5). ND96+ media was used for oocyte storage media (2.5mM sodium pyruvate and 6.7mM theophylline added). No gentamicin was added to the ND96+ storage media in order to avoid distorting ACh-induced responses (Amici, et al., 2005 (link)).

Marotta C.B., Lester H.A, & Dougherty D.A. (2015). An unaltered orthosteric site and a network of long-range allosteric interactions for PNU-120596 in α7 nicotinic acetylcholine receptors. Chemistry & biology, 22(8), 1063-1073.

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Unnatural Amino Acid Incorporation in Rat nAChR Subunits

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Rat nAChR α4 and β2 subunits
were in pAMV (unnatural mutagenesis) and pGEMhe (natural mutagenesis)
vectors. Site-directed mutagenesis was performed using the QuikChange
protocol (Stratagene). Circular DNA of α4 and β2 in pAMV
was linearized with the NotI restriction enzyme and the plasmids in
pGEMhe were linearized with the SbfI restriction enzyme. After purification
(Qiagen), the T7 mMessage Machine kit (Ambion) was used to in vitro transcribe mRNA from linearized DNA templates.
QIAGEN’s RNeasy RNA purification kit was used to isolate the
transcribed mRNA product.
For unnatural amino acid incorporation,
the amber (UAG) stop codon was used for all α4 subunit incorporation
and the opal (UGA) stop codon was used for the β2 subunit incorporation.
74-nucleotide THG73 tRNA (for UAG) and 74-nucleotide TQOpS’
tRNA (for UGA) were in vitro transcribed using the
MEGAshortscript T7 (Ambion) kit and isolated using Chroma Spin DEPC-H₂O columns (Clontech). Synthesized unnatural amino acids coupled
to the dinucleotide dCA were enzymatically ligated to the appropriate
74-nucleotide tRNA as previously described.^{13 (link),28}

Marotta C.B., Rreza I., Lester H.A, & Dougherty D.A. (2014). Selective Ligand Behaviors Provide New Insights into Agonist Activation of Nicotinic Acetylcholine Receptors. ACS Chemical Biology, 9(5), 1153-1159.

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Nonsense Suppression Method for ncAA Incorporation

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The nitroveratryloxycarbonyl-protected ncAA naphthalene esterified with 5′-O-phosphoryl-2′-deoxycytidylyl-(3′→5′)adenosine was incorporated via the nonsense suppression method (Dougherty and Van Arnam, 2014 (link)). Modified Tetrahymena thermophila tRNA was prepared by annealing full-length 5′ and 3′ DNA strands (Integrated DNA Technologies); RNA was synthetized using the T7-Scribe transcription kit (CELLSCRIPT) and purified with Chroma Spin DEPC-H₂O columns (Clontech). The ncAA naphthalene was ligated to the tRNA with T4 DNA ligase (New England Biolabs). The aminoacylated tRNA was purified with phenol-chloroform extraction and ethanol purification, air dried, and stored at −80°C until use. The pellet was resuspended in 1 μl water and deprotected with 400-W UV light (Newport UV lamp [66921], including Newport power supply [69920]) immediately before injection into oocytes. The deprotected aminoacylated tRNA was mixed 1:1 with mASIC1a mRNA containing a TAG codon at position 50 and injected into oocytes.

Sheikh Z.P., Wulf M., Friis S., Althaus M., Lynagh T, & Pless S.A. (2021). The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs. The Journal of General Physiology, 153(10), e202112899.

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Incorporation of NVOC-Protected ncAA into Protein

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The nitroveratryloxycarbonyl (NVOC) -protected non-canonical amino acid (ncAA) naphthalene esterified with 5'-O-phosphoryl-2'-deoxycytidylyl-(3'-->5')adenosine (pdCpA) was incorporated via the nonsense suppression method [30] . Modified Tetrahymena thermophila tRNA was prepared by annealing full-length 5' and 3' DNA strands (Integrated DNA Technologies, Belgium); RNA was synthetized using the T7-Scribe transcription kit (Cellscript) and purified with Chroma Spin DEPC-H2O columns (Clontech, CA, USA). The ncAA naphthalene was ligated to the tRNA with T4 DNA ligase (New England Biolabs, MA, USA). The aminoacylated tRNA was purified with phenol-chloroform extraction and ethanol purification, air dried and store at -80 % until use. The (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. pellet was resuspended in 1 uL water and deprotected with 400 W UV light (Newport UV lamp, # 66921, including Newport power supply # 69920) immediately before injection into oocytes. The deprotected aminoacylated-tRNA was mixed 1:1 with mASIC1a mRNA containing a TAG codon at position 50 and injected into oocytes.

Sheikh Z.P., Wulf M., Friis S., Althaus M., Lynagh T., & Pless S.A. (2021). The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs.

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