After reaction with glycogen synthase, the glycogen underwent two rounds of ethanol precipitation, as described above except that the reaction was diluted two-fold initially and dissolved in two times the reaction volume after the first precipitation. The glycogen was finally dissolved in 150-160 μl of 10 mM Tris-HCl pH 7.5. A portion (70 μl) was subjected to gel filtration on a spin column (Promega) containing ~1 ml of packed Sephadex G50 resin (Sigma) which had been equilibrated with 10 mM Tris-HCl pH 7.5 and extensively washed with the same buffer. Immediately prior to loading the samples, excess buffer was removed by centrifugation (1,000 x g for 1 min at 16°C). Sample (70 μl) was applied to the spin column, the column centrifuged (1,000 x g for 1 min at 16°C), and the flow-through collected. The gel filtered glycogen and an equivalent unfiltered aliquot of 70 μl were dried in a Speed Vac, dissolved in 25 μl of 10 mM Tris-HCl pH 7.5 and aliquots (20 μl) were made 1X in SDS loading buffer and subjected to SDS-PAGE. From quantitation of the 14C-labeled glycogen, recovery after gel filtration was ~95%.
Spin column
Manufactured by Promega
Sourced in United States
Spin columns are a type of laboratory equipment used for the purification and separation of various biomolecules, such as DNA, RNA, and proteins. They consist of a cylindrical column containing a porous matrix that allows the selective binding and elution of the target molecules. Spin columns rely on centrifugal force to facilitate the purification process, making them a versatile and efficient tool in many biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Sephadex g50 resin, by Merck Group (1 mentions)
Cell lysis solution, by Qiagen (1 mentions)
Nd 1000 spectrophotometer, by Avantor (1 mentions)
Precipitation solution, by Qiagen (1 mentions)
Supersciptii, by Thermo Fisher Scientific (1 mentions)
Mnase, by New England Biolabs (1 mentions)
Amicon ultra 0.5 centrifugal filter, by Merck Group (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Pureyield plasmid miniprep system, by Promega (1 mentions)
Escherichia coli jm109 competent cells, by Takara Bio (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Myiq system software version 1, by Bio-Rad (1 mentions)
Myiq single colour real time pcr detection system, by Bio-Rad (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Sephadex g 50, by Merck Group (1 mentions)
E coli dh5α competent cells, by Promega (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Myiq single color real time pcr detection system, by Bio-Rad (1 mentions)
10 protocols using spin column
1
Glycogen Purification and Analysis
2
Bacterial RNA Isolation and cDNA Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total bacterial RNA was isolated from bacteria grown under different growth conditions after killing by the addition of 0.2 volumes of 95% ethanol, 5% phenol, pH 4.3. Pellets were resuspended in 10 mM Tris, 1 mM EDTA containing 2 mg ml-1 lysozyme and incubated at 37°C for 30 min. Cell lysis solution (Qiagen, Hilden, Germany) was added and the mixture was incubated at 65°C for 5 min and at room temperature for 10 min. After the addition of precipitation solution (Qiagen, Hilden, Germany) and incubation on ice for 5 min cell debris, proteins and DNA were pelleted. The RNA containing supernatant was mixed with ethanol and loaded on a spin column (Promega). Further RNA purification and DNase digestion was done as described by the manufacturer.
A total of 50 μg of RNA of six separate experiments was reverse transcribed to cDNA and labelled with Cy3- or Cy5-conjugated dCTP (GE Healthcare) using reverse transcriptase (SupersciptII, Invitrogen) and random hexamers as primers. RNA was removed by hot-alkali treatment. Labelled cDNA was purified using a Qiaquick PCR purification kit and quantified by Nano-Drop analysis (ND-1000 Spectrophotometer, Peqlab).
A total of 50 μg of RNA of six separate experiments was reverse transcribed to cDNA and labelled with Cy3- or Cy5-conjugated dCTP (GE Healthcare) using reverse transcriptase (SupersciptII, Invitrogen) and random hexamers as primers. RNA was removed by hot-alkali treatment. Labelled cDNA was purified using a Qiaquick PCR purification kit and quantified by Nano-Drop analysis (ND-1000 Spectrophotometer, Peqlab).
3
Micrococcal Nuclease Assay for DNA Fragmentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reaction products were further analyzed by micrococcal nuclease (MNase) assays. First, the reaction buffer was exchanged to the CutSmart buffer (New England BioLabs; B7204) using a 30 K MWCO Amicon Ultra-0.5 Centrifugal Filter (Merck; UFC 503096). Then, we added 0.4 μl of 20 U/μl MNase (New England BioLabs; M0247S) to the reaction products and incubated them at room temperature for 30 and 60 min. The DNA fragments were purified using a spin column (Promega; A9281) and were run on e-PAGEL (ATTO; 2331830) in Tris-glycine buffer at 21 mA and 25°C for 75 min. The gels were stained with SYBR Gold Nucleic Acid Gel Stain, imaged using the iBright FL 1500 Imaging System and analyzed using Image J software (20 ).
4
Molecular Cloning and Sequencing of O. glaberrima
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PCR products were gel purified using a spin-column (Promega, Madison), ligated into the pGEM-T Easy Vector (Promega, Madison), and the ligated product was used to transform Escherichia coli JM109 competent cells (Takara, Japan) following the manufacturer’s instructions. The plasmid DNA of positive clones was extracted using the PureYield™ Plasmid Miniprep System (Promega, Madison), and their sequence determined using the pUC/M13 forward or gene-specific primers. The amplicon identity was confirmed by nucleotide similarity using the Basic Local Alignment Search Tool (BLAST) software.
The O. glaberrima (IRGC accession # 96717, variety name CG14) genome sequence was obtained from the Arizona Genomics Institute of the University of Arizona (ftp://glabgenome@ftp.genome.arizona.edu/ ), and local BLAST was performed using the program BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html , version 7.1.11) and/or gramene (http://www.gramene.org/ ). Sequence alignment was performed using the MAFFT 7 software (http://mafft.cbrc.jp/alignment/server/index.html ), and Jalview (http://www.jalview.org/ ).
The O. glaberrima (IRGC accession # 96717, variety name CG14) genome sequence was obtained from the Arizona Genomics Institute of the University of Arizona (
5
NISTmAb Reduction, Alkylation, and Tryptic Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NISTmAb (500
μg) was denatured at room temperature for 10 min using guanidine
hydrochloride (6 M) in 50 μL of 100 mmol/L Tris HCI (Tris(hydroxymethyl)aminomethane
hydrochloride) buffer (pH = 8). Reduction and alkylation were varied
as follows: (a) the sample was neither reduced nor alkylated or (b)
the sample was partially reduced by adding 5 μL of 200 mmol/L
TCEP to the above denatured mixture and incubating at room temperature
for 1 h, followed by alkylation with 10 μL of 200 mmol/L IAM
at room temperature in the dark for 1 h. The alkylated protein solution
was desalted with a Zeba spin column (7 K MWCO) prior to adding 10
μg of Promega trypsin. The resulting mixture was subjected to
digestion with sequencing-grade trypsin at 37 °C for 0.25, 2,
and 18 h.
μg) was denatured at room temperature for 10 min using guanidine
hydrochloride (6 M) in 50 μL of 100 mmol/L Tris HCI (Tris(hydroxymethyl)aminomethane
hydrochloride) buffer (pH = 8). Reduction and alkylation were varied
as follows: (a) the sample was neither reduced nor alkylated or (b)
the sample was partially reduced by adding 5 μL of 200 mmol/L
TCEP to the above denatured mixture and incubating at room temperature
for 1 h, followed by alkylation with 10 μL of 200 mmol/L IAM
at room temperature in the dark for 1 h. The alkylated protein solution
was desalted with a Zeba spin column (7 K MWCO) prior to adding 10
μg of Promega trypsin. The resulting mixture was subjected to
digestion with sequencing-grade trypsin at 37 °C for 0.25, 2,
and 18 h.
6
RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intestinal specimens suspended in RNA lysis buffer containing β-mercaptoethanol were homogenized using the TissueLyser (Qiagen, Hilden, Germany) for 1 minute/25 Hz and total RNA was isolated using spin columns based on manufacturer's instructions (Promega, Madison, WI, USA). RNA was reverse-transcribed to cDNA using iScriptTM cDNA Synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The PCR reaction mixture, containing iQSYBR Green Supermix (Bio-Rad Laboratories Inc.) was prepared based on manufacturer’s instructions and qRT-PCR analysis was performed using the MyiQ single-colour real time PCR detection system (Bio-Rad Laboratories Inc.) with MyiQ System Software Version 1.0.410 (Bio-Rad Laboratories Inc.). Commercially manufactured gene specific primers (Eurogentec, Seraing, Belgium) were used after confirmation of specificity and efficiency tests by qRT-PCR with dilution series of pooled cDNA at a temperature gradient (55°C to 65°C) for primer-annealing and subsequent melting curve analysis (S2 Table ). The mRNA quantity was calculated relative to the expression of β-Actin (ACTB) reference gene.
7
Radioactive Substrates for Enzyme Assays
8
HSP40 Gene Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three fragments (A, B, and C) (Figure 1 ) were separated based on the HSP40 sequence of T. gondii ME49 isolate (ToxoDB: TGME49_265310), and the amplifications were performed by PCR using three pairs of specific primers, respectively (Table 2 ). Thermal cycling conditions were according to the following protocol: initial denaturation at 94°C for 10 min followed by 35 cycles composing of 94°C for 1 min, 54.7°C (amplicon A), 66.8°C (amplicon B) or 64.0°C (amplicon C) for 45 s respectively, and 72°C for 2 min, and the additional extension step was carried out at 72°C for 10 min. Negative control without gDNA was included in each amplification. PCR amplifications were confirmed by agarose gel electrophoresis as previously described [15 (link)]. All the PCR products were purified using spin columns (Promega, Madison, USA), ligated with pMD18-T vector (TaKaRa, Dalian, China), and transformed into E. coli DH5α competent cells (Promega) according to the manufacturers' instructions. The positive colonies confirmed by PCR were sequenced by Shanghai Sangon Biological Engineering Biotechnology Company. All the experiments were run in triplicate.
9
Quantitative RT-PCR Analysis of IL-33 and ST2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression was determined by quantitative (q)RT-PCR, as described previously [13] . In brief, samples of the distal small intestine and lung tissue were homogenized in RNA lysis buffer with β-mercaptoethanol. RNA was extracted using spin columns according to the manufacturer's instructions (Promega, Madison, Wis., USA). cDNA was prepared from 1 μg of RNA, using the iScript TM cDNA synthesis kit ( Bio-Rad, Hercules, Calif., USA). qRT-PCR was performed using the MyIQ single-color real-time PCR detection system (Bio-Rad) with iQSYBR green supermix (Bio-Rad). IL-33 and ST2 primers were derived from the NCBI GenBank and manufactured commercially (Eurogentec, Seraing, Belgium); IL-33: forward 5 ′ -GGTGTGGATGGGAAGAAGCTG-3 ′ , reverse 5 ′ -GAGGACTTTTTGTGAAGGACG-3 ′ and ST2 forward 5 ′ -CAAGTAGGACCTGTGTGCCC-3 ′ and reverse: 5 ′ -CGT GTCCAACAATTGACCTG-3 ′ . The relative amounts of gene expression were standardized and calculated by the expression of house-keeping control gene (β-actin) as an internal standard, using the 2 -∆ ∆ Ct method.
10
Wasp DNA Extraction and Bisulfite Conversion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All four biological replicates (each consisting of 10 wasps) were washed with 70% ethanol, vacuum dried, and crushed in 100 mL of phosphate-buffered saline. For DNA isolation, we followed a modified protocol of Promega. In short, we added 100 mL of Nuclei Lysis Solution (Promega) and 2 mL of Proteinase K (20 mg/mL; Roche) and incubated the lysate for 60 min at 607C. After incubation, we added 170 mL of Lysis Buffer (Promega), vortexed shortly, and centrifuged the mixture for 10 min at 14,000 rpm. We used spin columns and DNA wash buffer (Promega) to isolate the DNA from the supernatant. For each sample, an aliquot of DNA was converted with bisulfite following the protocol of the EZ DNA Methylation-Gold kit (Zymo).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!