A total of 300 inseminated chicken eggs at embryonic E9 were obtained from a native farm and artificially incubated at 37.5°C and 60% relative humidity. Eggs were randomly assigned to 2 groups, control and betaine group. On E11, betaine (B2629, Sigma–Aldrich, St Louis, MO) dissolved in saline was injected into the yolk sac at the dose of 2.5 mg per egg in a volume of 100 μL. The dose was determined according to the range of betaine concentrations in chicken eggs reported previously (Zeisel et al., 2003 (link), Hu et al., 2015 ; Idriss et al., 2017 (link)). The incubator was set according to our previous publications. On E19, female embryos were identified and removed from the eggs, euthanized by decapitation which is approved by the American Veterinary Medical Association Guidelines for the Euthanasia of Animals: 2013 Edition. Then blood samples were taken, and plasma was separated and stored at −20°C. Adrenal samples were collected, rapidly frozen in liquid nitrogen and stored at −80°C for further analysis.
Betaine
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Macao, Sao Tome and Principe, Japan, Canada, Denmark, Malaysia
Betaine is a naturally occurring organic compound that functions as an osmolyte, helping to maintain the structural integrity of proteins in cells. It is commonly used in biochemical and biological laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bst dna polymerase large fragment, by New England Biolabs (31 mentions)
Mgso4, by New England Biolabs (31 mentions)
Bst dna polymerase, by New England Biolabs (23 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (20 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (16 mentions)
Mgso4, by Merck Group (15 mentions)
Dntps, by Thermo Fisher Scientific (14 mentions)
Mgcl2, by Merck Group (10 mentions)
Dntps, by New England Biolabs (9 mentions)
Proline, by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Mgcl2, by Thermo Fisher Scientific (8 mentions)
Lactic acid, by Merck Group (8 mentions)
Tween 20, by Merck Group (8 mentions)
Choline chloride, by Merck Group (8 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (8 mentions)
Isothermal amplification buffer, by New England Biolabs (7 mentions)
Qiaprep spin miniprep kit, by Qiagen (7 mentions)
Thermopol reaction buffer, by New England Biolabs (7 mentions)
Thermopol buffer, by New England Biolabs (6 mentions)
318 protocols using betaine
1
Betaine Supplementation in Chicken Embryos
2
Dietary Betaine Modulates HFD-Induced Obesity
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All animal studies were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. 40 Kunming mice (female, six weeks old, 19–20 g) were treated with a high-fat diet (HFD: 40.4% fat, 14.6% protein, 45.2% carbohydrate by energy) or normal chow (NCW: 13.2% fat, 23.2% protein, 63.6% carbohydrate by energy) for 13 weeks, respectively. Additionally, mice were housed at 22–24 °C and given free access to water, under controlled conditions of light and temperature. To evaluate the effect of betaine on HFD-induced obesity and hyperglycemia, 12 HFD- and 12 NCW-fed mice were treated with or without 1% (weight/volume, W/V) betaine (Sigma, St. Louis, MO, USA) in water according to a previous report [32 (link)]. Moreover, diabetes induced in female Kunming mice fed a HFD for 30 days was treated by intraperitoneal injection with 200 mg/kg streptozotocin [33 (link)] (STZ; Sigma-Aldrich, St. Louis, MO, USA). Blood glucose levels were measured using an Accu Check Advantage Glucometer (Roche, Dublin, Ireland, cat.06583261001).
3
Real-Time Isothermal Detection of Pseudoalteromonas lurida
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The P. lurida RealAmp reaction system based on the gyrB gene was as follows: 2 mM of MgSO4, 0.4 mM of dNTP, 1.2 μM each of Q-FIP and Q-BIP, 0.2 μM each of Q-F3 and Q-B3, 0.4 μM each of Q-LF and Q-LB, 0.2 M of betaine (Sigma-Aldrich, MO, USA), 2.5 μL of 10× ThermoPol Buffer, 8 U of Bst polymerase (New England Biolabs Inc., Ipswich, Massachusetts, USA), 1/300 dilution of 0.3 μL of 10,000× SYBR green I, 1 μL of DNA template, and added sterilized distilled water up to a final volume of 25 μL.
The P. lurida RealAmp reaction system based on the aprX gene was as follows: 2 mM of MgSO4, 0.4 mM of dNTP, 1.4 μM each of L-FIP and L-BIP, 0.28 μM each of L-F3 and L-B3, 0.28 μM each of L-LF and L-LB, 0.2 M of betaine (Sigma-Aldrich), 2.5 μL of 10× ThermoPol Buffer, 8 U of Bst polymerase (New England Biolabs Inc.), 1/300 of dilution of 0.3 μL of 10,000× SYBR green I, 1 μL of DNA template, and added sterilized distilled water up to a final volume of 25 μL.
Further, 20 μL of mineral oil was used to cover the reaction system to prevent contamination. For the gyrB and aprX genes, reaction tubes were held at 61°C and 62°C, respectively, for 40 min in the fluorescent quantitative PCR instrument (QuantStudio 3, Applied Biosystems, Waltham, MA, USA). After the RealAmp reaction was complete, the detection result was determined based on the peak time.
The P. lurida RealAmp reaction system based on the aprX gene was as follows: 2 mM of MgSO4, 0.4 mM of dNTP, 1.4 μM each of L-FIP and L-BIP, 0.28 μM each of L-F3 and L-B3, 0.28 μM each of L-LF and L-LB, 0.2 M of betaine (Sigma-Aldrich), 2.5 μL of 10× ThermoPol Buffer, 8 U of Bst polymerase (New England Biolabs Inc.), 1/300 of dilution of 0.3 μL of 10,000× SYBR green I, 1 μL of DNA template, and added sterilized distilled water up to a final volume of 25 μL.
Further, 20 μL of mineral oil was used to cover the reaction system to prevent contamination. For the gyrB and aprX genes, reaction tubes were held at 61°C and 62°C, respectively, for 40 min in the fluorescent quantitative PCR instrument (QuantStudio 3, Applied Biosystems, Waltham, MA, USA). After the RealAmp reaction was complete, the detection result was determined based on the peak time.
4
Optimizing RT Reaction Efficiency
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In order to improve the efficiency of the RT, we tested the addition of reaction enhancers, including MgCl2, betaine, trehalose, and polyethylene glycol (PEG 8000). The final reaction volume of 10 µl was maintained by adjusting the volume of H2O.
For this, we added increasing concentrations of MgCl2 (3, 6, 9, and 12 mM; Sigma-Aldrich) in the RT buffer in the presence or absence of 1 M betaine (Sigma-Aldrich). Furthermore, the addition of 1 M betaine and 0.6 M trehalose (Sigma-Aldrich) was compared to the standard RT protocol. Lastly, increasing concentrations of PEG 8000 (0, 3, 6, 9, 12, and 15% W/V) were also tested.
For this, we added increasing concentrations of MgCl2 (3, 6, 9, and 12 mM; Sigma-Aldrich) in the RT buffer in the presence or absence of 1 M betaine (Sigma-Aldrich). Furthermore, the addition of 1 M betaine and 0.6 M trehalose (Sigma-Aldrich) was compared to the standard RT protocol. Lastly, increasing concentrations of PEG 8000 (0, 3, 6, 9, 12, and 15% W/V) were also tested.
5
Optimization of PSR Assay for Genetic Analysis
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The PSR assay was done using a temperature gradient of 60–69°C and reaction time of 60–90 min. The primer concentration was also optimized. The reaction mixture comprised of 2.5 µl of 10X Thermopol reaction buffer (New England Biolabs, Massachusetts, United States) containing 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Tween 20, 10–40 µM each forward and reverse primer, 1.4 mM dNTP mix (Thermo Fisher Scientific), 0.8 M Betaine (Merck), 6 mM MgSO4 (New England Biolabs), 8–16 U of Bst DNA polymerase large fragment (New England Biolabs), 50 ng DNA template, and the final volume was adjusted to 25 µl with sterile distilled water. No-template water control (NTC) was used with each run.
Restriction digestion of PSR products was done using 5 µl of PSR product, 2 µl of NcoI FastDigest enzyme (Thermo Fisher Scientific), 2 µl 10X FastDigest Green Buffer (Thermo Fisher Scientific), in a final volume of 20 µl for 1 h at 37°C. The digested products were resolved in 2% agarose gel electrophoresis as described above. Based on the results of PSR, primer pair AG339F-AG340R was further assessed for cross-reactivity and sensitivity.
Restriction digestion of PSR products was done using 5 µl of PSR product, 2 µl of NcoI FastDigest enzyme (Thermo Fisher Scientific), 2 µl 10X FastDigest Green Buffer (Thermo Fisher Scientific), in a final volume of 20 µl for 1 h at 37°C. The digested products were resolved in 2% agarose gel electrophoresis as described above. Based on the results of PSR, primer pair AG339F-AG340R was further assessed for cross-reactivity and sensitivity.
6
Succinic Semialdehyde Dehydrogenase Assay
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Succinic semialdehyde (SSA), succinic acid (SA), oxidized (NAD+) and reduced (NADH) nicotinamide adenine dinucleotide, isopropyl-β-d-thiogalactopyranoside (IPTG), phenylmethylsulfonyl fluoride (PMSF), poly-L-lysine, betaine and SigmaFast inhibitor cocktail were purchased from Merck (Darmstadt, Germany). Anti-SSADH (cat. sc-390754) and anti-His antibodies (cat. sc-8036) were from Santa Cruz Biotechnology (Heidelberg, Germany). ROTI®Mount FluorCare DAPI and 2-mercaptoethanol were from Roth (Karlsruhe, Germany). Hygromycin B was from Applichem GmbH (Darmstadt, Germany), and MACSfectinTM transfection reagent was from Miltenyi Biotech (Bergisch Gladbach, Germany). Puromycin, zeocin, Alexa Fluor 488-conjugated secondary anti-rabbit antibody, and Mitotracker Orange were from Invitrogen/Thermo Fischer Scientific GmbH (Darmstadt, Germany). Further chemicals were of the highest purity available.
7
Lycium Fruit Metabolite Profiling
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Deuterium oxide (D2O, 99.98%), maleic acid, and succinic acid were obtained from Sigma–Aldrich (Milwaukee, WI, US). The reference compounds (betaine, citric acid, proline, threonine, and alanine) were purchased from Merck (Darmstadt, Germany). The ultrapure water (H2O) was prepared with Milli-Q water purification system (Millipore, Bedford, MA, US). The analytical cartridge column was using Thermo Fisher Scientific (Waltham, MA, US) HyperSep SCX strong cation exchanger SPE columns (2000 mg). Lycium fruit samples 1–23 and 27–42 were purchased from the markets in China. Samples 24–26 were collected in Shanxi province of China in Oct, 2009. All samples were purchased and collected by Dr. Yong Peng. The materials were identified by Prof. C. S. Kuoh (Department of Life Science, National Cheng Kung University), and voucher specimen (TSWu 20100708-01-42) have been deposited in the Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
8
CYP Genotyping for Anticonvulsant Response
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DNA was extracted from peripheral blood by salting-out [28 (link)]. SNPs characterizing CYP2C19*2, CYP2C19*3, CYP2C19*17, CYP2C9*2, CYP2C9*3 and CYP3A5*3 alleles (Table 1 ) were genotyped using TaqMan allele-specific polymerase chain reactions (Life Technologies). Amplification reactions were as follow: TaqMan PCR Mastermix 1x/μL, TaqMan SNP genotyping assay 1x/μL, genomic DNA 1ng/μL, ultrapure water to complete 7μL volume. rs12248560 required 500mM of Betaine (Sigma Aldrich) in the final reaction.
Allele discrimination was evaluated in a Line Gene 9600 (BIOER Technology CO.) comparing amplification curves and fluorescence levels before and after amplification (45 cycles of 15 seconds at 95°C and 1min at 60°C).
The candidate CYP genes were chosen according to the described association to the aromatic anticonvulsant-reactions [14 (link),15 (link),18 ,19 (link),29 (link)] in different populations and the importance in the metabolism of these drugs (Table 1 ).
According to the alleles presented, each subject was classified with normal (EM), decrease (IM/PM) or increased (UM) function for each enzyme (Table 2 ).
Allele discrimination was evaluated in a Line Gene 9600 (BIOER Technology CO.) comparing amplification curves and fluorescence levels before and after amplification (45 cycles of 15 seconds at 95°C and 1min at 60°C).
The candidate CYP genes were chosen according to the described association to the aromatic anticonvulsant-reactions [14 (link),15 (link),18 ,19 (link),29 (link)] in different populations and the importance in the metabolism of these drugs (
According to the alleles presented, each subject was classified with normal (EM), decrease (IM/PM) or increased (UM) function for each enzyme (
9
Development and Validation of LAMP Assay for Mink Behavior Detection
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Based on the candidate fragment of RAPD marker A10, six primers (Outer primers F3 and B3, forward inner primer FIP, reverse inner primer BIP, and loop primers LF and LB) were designed by PrimerExplorer4 (Fujitsu, Tokyo, Japan; http://primerexplorer.jp/e/). The location and sequence of each primer are summarized in Table 1.
LAMP was performed in a total 25 mL reaction mixture containing 0.8 mM each of FIP and BIP, 0.2 mM each of the outer primers, 0.4mM each of LF and LB, 1.2 mM dNTPs, 1.0 M betaine (Sigma, St. Louis, MO), 8 mM MgSO 4 , 1 µL Bst DNA polymerase (New England Biolabs, Beverly, US) and 2 µL the extracted template DNA (Nagamine et al. 2001 , Nagamine et al. 2002a , Fukuta et al. 2003 , Gunimaladevi et al. 2004 ). The reaction temperature was optimized and LAMP was carried out for 40 min and terminated at 80C for 2 min. LAMP products were subjected to electrophoresis on a 2% agarose gel. Healthy and stereotype behavior individuals (15 of each) from another mink farm were tested by SCAR and LAMP, and sensitivity of the detection was compared between these two methods. A c 2 test in a 2×2 table was performed to determine whether a relationship exists between the SCAR and LAMP markers of the two groups. SAS8.0 software was used to analyze these data.
LAMP was performed in a total 25 mL reaction mixture containing 0.8 mM each of FIP and BIP, 0.2 mM each of the outer primers, 0.4mM each of LF and LB, 1.2 mM dNTPs, 1.0 M betaine (Sigma, St. Louis, MO), 8 mM MgSO 4 , 1 µL Bst DNA polymerase (New England Biolabs, Beverly, US) and 2 µL the extracted template DNA (Nagamine et al. 2001 , Nagamine et al. 2002a , Fukuta et al. 2003 , Gunimaladevi et al. 2004 ). The reaction temperature was optimized and LAMP was carried out for 40 min and terminated at 80C for 2 min. LAMP products were subjected to electrophoresis on a 2% agarose gel. Healthy and stereotype behavior individuals (15 of each) from another mink farm were tested by SCAR and LAMP, and sensitivity of the detection was compared between these two methods. A c 2 test in a 2×2 table was performed to determine whether a relationship exists between the SCAR and LAMP markers of the two groups. SAS8.0 software was used to analyze these data.
10
Betaine and LPS Effects on Intestinal Barrier
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Betaine and LPS were purchased from Sigma-Aldrich (Saint Louis, USA) and dissolved in PBS to prepare stock solutions with concentrations of 100 mmol/L and 1mg/mL respectively. Occludin (Catalog number: #91131) and Claudin-1 (Catalog number: #13995) were obtained from Cell Signaling Technology (Shanghai, China.). All the reagents were stored at -20℃.
Cell culture IPEC-J2 cells were obtained from Animal Nutrition & Human Health Laboratory, Hunan Normal University. IPEC-J2 cells were cultured in DMEM with 10% of FBS and 1% of penicillin-streptomycin (Hyclone, Logan, UT, USA) at 37 °C, 5% of CO 2 . The medium was refreshed every second day.
Cell culture IPEC-J2 cells were obtained from Animal Nutrition & Human Health Laboratory, Hunan Normal University. IPEC-J2 cells were cultured in DMEM with 10% of FBS and 1% of penicillin-streptomycin (Hyclone, Logan, UT, USA) at 37 °C, 5% of CO 2 . The medium was refreshed every second day.
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