Four polymorphisms (-216g / T,-191C / A, intron 1 (CA) n, and 497G / where can I purchase soma online South Dakota A) in the EGFR gene, the CYP3A4 * 1B, CYP3A5 * 3 and six polymorphisms (-15994G / A,-15622C / T, intron 1 16702G / A, intron 1 1143C / T, 421C / D and 34G / A) in the ABCG2 gene were genotyped. The polymerase chain reaction (PCR) was where can I purchase soma online South Dakota performed to amplify DNA sequences containing the polymorphisms of interest. Temperatures DNA sequences related genotype and annealing are listed in Appendix Table A2. For-191C / A and 216 g / T, CRP was created in a volume of 40 l with 3 mM MgCl 2, 1xQ-solution (Qiagen, Santa Clarita, where can I purchase soma online South Dakota Calif.), 100 mM of each dNTP, 125 nM forward and back primers, Taq DNA polymerase unit Hotstart (Qiagen), and 25 ng of DNA.
The reactions were denatured initially at 98 ° C for 10 minutes, then cycle 35 times at 98 ° C for 15 seconds, annealing at 62 ° C for 15 seconds and 72 ° C for 20 seconds. The PCR products were then purified and sequenced directly as described.where can I purchase soma online South Dakota 1 For other polymorphisms, PCR was performed in a volume of 15 l with 125 nM of each primer of PCR amplified two, 30 ng of genomic DNA, 2.5 mM MgCl 2, 100 M of each dNTP, where can I purchase soma online South Dakota and 0.375 U Qgold AmpliTa polymerase (Applied Biosystems, Foster City, CA) in a buffer provided by the manufacturer. Duplex PCR was used to amplify the ABCG2 polymorphisms.
Genotype EGFR intron 1 (CA) n polymorphism was performed as described.2 other polymorphisms, EGFR 497G / A, CYP3A4 * 1B, CYP3A5 where can I purchase soma online South Dakota * 3, and ABCG2 polymorphisms were genotyped using single base extension and distortion liquid chromatography, high performance (DHPLC). Briefly, PCR-amplified products were where can I purchase soma online South Dakota purified by treatment with shrimp alkaline phosphatase (Roche, Neuilly-sur-Seine, France) and exonuclease I (USB) at 37 ° C for 45 minutes before the SBE reaction. SBE reactions were performed in 12.6 s with 1 M of each SBE primer, 250 M each of the four ddNTP, 7.2 l of purified PCR product and 1.5 U ThermoSequenase (Amersham Pharmacia Biotech) in 1x reaction buffer where can I purchase soma online South Dakota provided by the manufacturer.
The reactions were performed in a thermal cycler 9600 (Applied Biosystems) under the following conditions: 96 ° C for 2 minutes, followed by 60 cycles of 96 ° C for 30 s, 55 ° C for 30 s and 60 ° C for 30 s. Wave 3500HT DHPLC system (TransgenomicInc.) was used to separate the products of the SBE. Before running the DHPLC, samples were denatured at 96 ° C for 4 minutes and stored at 4 ° C. For the analysis of DHPLC where can I purchase soma online South Dakota in the oven SBE, SBE products 8 l of each sample was injected. We used 'mutation detection "type of application as a template, select" Normal "where can I purchase soma online South Dakota own the type of cleaning (100% buffer B after each injection where can I purchase soma online South Dakota cleaning step), and manually set the following variables for this application: The flow rate was set 1.5 mL / min using a high performance column, the where can I purchase soma online South Dakota oven temperature was set at 70 where can I purchase soma online South Dakota ° C, and the gradient used for elution of the SBE products was 24% to 36.5% buffer B over 2.5 minutes (buffer B containing 25% acetonitrile).
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