EV-A71 genomic RNA was extracted using QIAamp Viral RNA mini kit (QIAGEN, Germany) according to the manufacturer’s instructions. EV-A71 cDNA was synthesized using pEV71-R (S1 Table ) using Superscript III reverse transcriptase (Invitrogen, USA). PCR was performed using pEV71-F and pEV71-R (S1 Table ) using Q5 High-Fidelity DNA polymerase (NEB, USA). The resulting 7.4 kbp product was cloned into pCR-XL-TOPO vector and transformed into XL10-GOLD ultracompetent cells (Agilent Technologies, USA). The internal T7 promoter of pCR XL TOPO was removed using deletion PCR followed by T4 DNA ligase-T4 polynucleotide kinase-DpnI (NEB, USA) treatment prior to transformation into XL10-GOLD ultracompetent cells (Agilent Technologies, USA). The resulting pT7-EV71 infectious clone was subjected to full-genome sequencing.
Xl10 gold ultracompetent cells
Manufactured by Agilent Technologies
Sourced in United States, Switzerland
XL10-Gold ultracompetent cells are a type of chemically competent bacterial cells used in molecular biology and genetic engineering applications. They are designed to facilitate the transformation of DNA into Escherichia coli (E. coli) bacteria, enabling the introduction of plasmids or other genetic material for various experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (6 mentions)
T4 dna ligase, by New England Biolabs (6 mentions)
Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (5 mentions)
Dpni, by New England Biolabs (4 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (4 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (4 mentions)
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Pureyield plasmid miniprep system, by Promega (3 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (3 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (3 mentions)
Midiprep kit, by Qiagen (3 mentions)
Sanger sequencing, by Genewiz (3 mentions)
E coli bl21 de3 star competent cells, by Thermo Fisher Scientific (3 mentions)
Quick ligation kit, by New England Biolabs (2 mentions)
Maxiprep, by Qiagen (2 mentions)
Pool oligonucleotides, by Integrated DNA Technologies (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Ptracer, by Thermo Fisher Scientific (2 mentions)
Gibson reaction master mix, by New England Biolabs (2 mentions)
83 protocols using xl10 gold ultracompetent cells
1
Construction of EV-A71 Infectious Clone
2
Pooled Oligonucleotide Library Construction
3
Rapid Scaffold ssDNA Production
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The 7560 nt single-stranded scaffold
strand was produced as described
in literature.21 (link),63 (link) In short, 1 μL of 100 nM
ssDNA was transformed in 90 μL XL10-Gold Ultracompetent cells
(Agilent) and grown overnight at 37 °C on agar plates supplemented
with tetracycline (10 μg/mL, Sigma-Aldrich) according to the
manufacturer’s protocol. A plaque was used to inoculate 300
mL of 2xYT medium (16 g/L peptone, 5 g/L NaCl, 10 g/L yeast extract)
supplemented with 5 mM MgCl2, and the culture was incubated
for 4 h at 37 °C. The cells were pelleted by centrifugation,
and the bacteriophages were extracted from the supernatant by PEG
fractionation.63 (link) After centrifugation,
the pellet was reconstituted in TE buffer (10 mM Tris, 1 mM EDTA,
pH 8.5) and lysed using buffers P2 and P3 (Qiagen). After ethanol
precipitation, the single-stranded phage DNA was reconstituted in
TE buffer and stored at −30 °C in DNA LoBind tubes (Eppendorf).
The concentration was determined by measuring the absorption at 260
nm (ND-1000, Thermo Scientific) and the respective extinction coefficient
(ssDNA 7560: 7.43 × 10–1 cm–1).
strand was produced as described
in literature.21 (link),63 (link) In short, 1 μL of 100 nM
ssDNA was transformed in 90 μL XL10-Gold Ultracompetent cells
(Agilent) and grown overnight at 37 °C on agar plates supplemented
with tetracycline (10 μg/mL, Sigma-Aldrich) according to the
manufacturer’s protocol. A plaque was used to inoculate 300
mL of 2xYT medium (16 g/L peptone, 5 g/L NaCl, 10 g/L yeast extract)
supplemented with 5 mM MgCl2, and the culture was incubated
for 4 h at 37 °C. The cells were pelleted by centrifugation,
and the bacteriophages were extracted from the supernatant by PEG
fractionation.63 (link) After centrifugation,
the pellet was reconstituted in TE buffer (10 mM Tris, 1 mM EDTA,
pH 8.5) and lysed using buffers P2 and P3 (Qiagen). After ethanol
precipitation, the single-stranded phage DNA was reconstituted in
TE buffer and stored at −30 °C in DNA LoBind tubes (Eppendorf).
The concentration was determined by measuring the absorption at 260
nm (ND-1000, Thermo Scientific) and the respective extinction coefficient
(ssDNA 7560: 7.43 × 10–1 cm–1).
4
Generation of Cysteine-Free Zmpste24 Variant
5
Site-Directed Mutagenesis of Fv1 and Fv7 Genes
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A PCR based strategy was used to introduce site directed changes to the
Fv1 or Fv7 genes. 10 ng of plasmid
carrying the gene was used together with 150 ng of each primer containing the
altered sequence and spanning the site to be mutated. The reaction was performed
using PfuUltra (Agilent) with 18 cycles of denaturation at 95°C for 30 seconds,
55°C for 1 minute and 68°C for 9 minutes 30 seconds. The reaction mixture was
then digested with DpnI (New England BioLabs) for 1 hour before using 4 μl for
the transformation of XL10 gold ultracompetent cells (Agilent). Colonies were
screened for the mutation and verified by sequencing.
Fv1 or Fv7 genes. 10 ng of plasmid
carrying the gene was used together with 150 ng of each primer containing the
altered sequence and spanning the site to be mutated. The reaction was performed
using PfuUltra (Agilent) with 18 cycles of denaturation at 95°C for 30 seconds,
55°C for 1 minute and 68°C for 9 minutes 30 seconds. The reaction mixture was
then digested with DpnI (New England BioLabs) for 1 hour before using 4 μl for
the transformation of XL10 gold ultracompetent cells (Agilent). Colonies were
screened for the mutation and verified by sequencing.
6
Site-Directed Mutagenesis of ImpA
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The pET15b plasmid containing the
wild type ImpA was used for mutagenesis. Forward primers and their
reverse complements were designed to replace specific residue positions
as described inTable 1 . Site directed mutagenesis for each mutation followed the same protocol
and was performed using the QuikChange Lightning site-directed mutagenesis
kit (Agilent Technologies) as per manufacturers protocol and transformed
to XL-10 Gold Ultracompetent Cells (Agilent Technologies). Plasmids
were extracted and transformed into BL21 DE3 PlysS competent cells
for protein expression. Each mutation was confirmed by sequencing.
wild type ImpA was used for mutagenesis. Forward primers and their
reverse complements were designed to replace specific residue positions
as described in
and was performed using the QuikChange Lightning site-directed mutagenesis
kit (Agilent Technologies) as per manufacturers protocol and transformed
to XL-10 Gold Ultracompetent Cells (Agilent Technologies). Plasmids
were extracted and transformed into BL21 DE3 PlysS competent cells
for protein expression. Each mutation was confirmed by sequencing.
7
CFTR Minigene Plasmid Generation
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Expression minigene plasmids were created as previously described [32 (link)]. CFTR-EMGi1-i5 contained abridged intron 1 (216 bp of 5′ and 212 bp of 3′), abridged intron 2 (311 bp of 5′ and 264 bp of 3′), abridged intron 3 (374 bp of 5′ and 456 bp of 3′), abridged intron 4 (307 bp of 5′ and 333 bp of 3′), and full-length intron 5 (882 bp). E60X, L88X, and Y122X variants were individually introduced to EMG plasmids through site-directed mutagenesis (SDM) as previously described [32 (link)]. Mutagenic primers were designed using QuickChange Primer Design tool. Primers were used to PCR-amplify EMG plasmid, followed by digest with DpnI, transformation of XL10-Gold ultracompetent cells (Agilent, Cedar Creek, TX, USA), and selection of colonies on LB-Ampicillin plates (Quality Biologicals, Gaithersburg, MD, USA). DNA minipreps were prepared (Denville Spinsmart Plasmid Miniprep DNA Purification Kit, Swedesboro, NJ, USA) and presence of the variant of interest was confirmed by Sanger sequencing. Selected miniprepped plasmid was used to transform XL10-Gold ultracompetent cells, and DNA maxipreps were prepared (Qiagen Plasmid Plus Maxi Kit, Hilden, Germany). Sanger sequencing was used to verify sequence of entire plasmid and confirm presence of the variant of interest.
8
Site-directed mutagenesis of amicyanin
9
Generation of Methylation-Resistant Actin Mutants
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For generation of methylation-resistant mutant actin constructs, the mCherry–actin-7 plasmid (Addgene plasmid no. 54966) was used. Mutagenesis was performed using the QuikChange II XL site-directed mutagenesis kit (Agilent), as per the manufacturer’s instructions. The following primers were used to generate the mCherry-ACTB-K68R/A mutants:
K68R, 5′-gctcgatggggtacctcagggtgaggatg-3′ (reverse) and 5′-catcctcaccctgaggtaccccatcgagc-3′ (forward); and K68A, 5′-tgctcgatggggtacgccagggtgaggatgcc-3′ (reverse) and 5′-ggcatcctcaccctggcgtaccccatcgagca-3′ (forward).
Mutagenized constructs were transformed into XL10-Gold Ultracompetent cells (Agilent), prepared using QIAprep Spin Miniprep Kits (Qiagen) and E.Z.N.A. Plasmid Maxi Kits (Omega Bio-tek), and transfected into HEK293T cells, as described above.
K68R, 5′-gctcgatggggtacctcagggtgaggatg-3′ (reverse) and 5′-catcctcaccctgaggtaccccatcgagc-3′ (forward); and K68A, 5′-tgctcgatggggtacgccagggtgaggatgcc-3′ (reverse) and 5′-ggcatcctcaccctggcgtaccccatcgagca-3′ (forward).
Mutagenized constructs were transformed into XL10-Gold Ultracompetent cells (Agilent), prepared using QIAprep Spin Miniprep Kits (Qiagen) and E.Z.N.A. Plasmid Maxi Kits (Omega Bio-tek), and transfected into HEK293T cells, as described above.
10
Enhancing Expression and Minimizing Recombination in Plasmid Constructs
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A modified pCaSpeR plasmid containing an actin5C promoter and a PhiC31-Integrase attB site was kindly provided by C. Alexandre and further modified as described next. To enhance expression and avoid positional effects, gypsy insulators were amplified from pVALIUM2024 adding 5’ EcoRI and XhoI, and 3’ BamHI and NheI restriction sites: the gypsy PCR product digested with EcoRI and NheI was cloned into identical sites in the modified pCaSpeR, making act5C-gypsy1; the gypsy PCR product digested with XhoI and BamHI was cloned into identical sites in act5C-gypsy1, making act5C-gypsy2. To minimise recombination, this plasmid as well as its FOFO derivatives were best grown in XL10-Gold Ultracompetent Cells (Agilent Technologies, Cat. No. 200314) at 30°C at 150 rpm.
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