Chloroform and 2-propanol were purchased from Roth (Karlsruhe, Germany) and methanol from Merck (Darmstadt, Germany). All solvents were HPLC grade. Nuclease-free water was obtained from B. Braun (Melsungen, Germany). Ammonium formate, sodium dodecyl sulfate (SDS), and cholesteryl ester (CE) standards were purchased from Sigma-Aldrich (Taufkirchen, Germany). Moreover, [25,26,26,26,27,27,27-D7]-cholesterol was acquired from Cambridge Isotope Laboratories (Andover, MA, USA) with isotope purity higher than 98%. Triglyceride (TG) and diglyceride (DG) standards were purchased from Larodan (Solna, Sweden). Phosphatidylcholine (PC), ceramide (Cer), sphingomyelin (SM), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) standards were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The composition of the added internal standard mixture is depicted in Table 1 .
Nuclease free water
Manufactured by B. Braun
Sourced in Germany
Nuclease-free water is a high-quality laboratory water product designed for use in sensitive molecular biology applications. It is treated to remove RNase, DNase, and other nucleases, ensuring the purity and integrity of nucleic acid samples. The product is produced and packaged under controlled conditions to minimize the risk of contamination.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Ammonium formate, by Merck Group (2 mentions)
Chloroform, by Carl Roth (2 mentions)
2 propanol, by Carl Roth (2 mentions)
Cholesteryl ester, by Merck Group (1 mentions)
Triglyceride tg, by Larodan (1 mentions)
Diglyceride dg, by Larodan (1 mentions)
Lysophosphatidylethanolamine lpe, by Avanti Polar Lipids (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions)
3 protocols using nuclease free water
1
Lipid Extraction and Quantification
2
Standardized Lipid Extraction and Analysis
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Chloroform and 2-propanol were purchased from Roth (Karlsruhe, Germany) and methanol from Merck (Darmstadt, Germany). All solvents were HPLC grade. Nuclease-free water was obtained from B. Braun (Melsungen, Germany). Ammonium formate, SDS, and CE standards were purchased from Sigma-Aldrich (Taufkirchen, Germany). [25,26,26,26,27,27,27-D7]-cholesterol was acquired from Cambridge Isotope Laboratories (Andover, MA, USA) with isotope purity higher than 98%. TG and DG standards were purchased from Larodan (Solna, Sweden). PC, Cer, SM, LPC, and LPE standards were purchased from Avanti Polar Lipids (Alabaster, AL, USA)
3
Validating Melanoma-Associated Variants
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All 10 variants were confirmed by Sanger sequencing using DNA from the two family members subjected to WES (II.2 and III.3, see Figure, Supplemental Digital Content 1, http://links.lww.com/MR/A177). Briefly, 20-100 ng of DNA was amplified through a touchdown PCR using the Platinum Taq DNA Polymerase following the manufacturer's instructions (Invitrogen, Carlsbad, California, USA). The PCR product was cleaned-up using the NucleoSpin Gel and PCR Clean-Up (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to manufacturer's instructions. Then, Sanger sequencing was performed using 20-50 ng of purified DNA mixed with 1 μl of 10 μM of sequencing primer and nuclease-free water (B. Braun, Melsungen, Germany) up to 10 μl. Later on, we used the same approach to evaluate the co-segregation of these variants with melanoma in all affected family members (see Figure, Supplemental Digital Content 2, http://links.lww.com/MR/A178 and Fig. 2)
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