《Table 1 Characteristics of RFLP-PCR primers, restriction endonucleases and product size》

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《TNF-α gene polymorphisms: association with age-related macular degeneration in Russian population》

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Polymorphisms were detected by polymerase chain reaction（PCR）followed by the restriction fragment length polymorphism（RFLP）method.PCR was carried out using a MyCycler?Thermal Cycler System（Bio Rad，USA）.The 20μL reaction solution contained one unit of TaqDNA polymerase（SibEnzyme，Novosibirsk，Russia），0.5μmol/L of each primer，0.25 mmol/L of each desoxynucleoside-triphosphate，and 50-200 ng of genomic DNA.Reaction buffer added to DNA polymerase contained 60 mmol/L of Tris-HCl（pH8.5，25℃），1.5 mmol/L MgCl2，25 mmol/L KCl，10 mmol/L2-mercaptoethanol，and 0.1%Triton X-100.Applied TNF PCR program was at 95℃for 5min，followed by 40 cycles of denaturation at 95℃for 30s，primer annealing at 59℃for 30s，and process extension at 72℃for 40s.Final extension step was carried out at 72℃for 3min.Amplification products were exposed to respective restriction enzymes（1-2 activity units）in20μL volume.After enzymatic digestion PCR products were size separated and visualized in 2.5%agarose gel.Specific primers used for amplification of TNF-αpromoter region at position-238G/A，-308G/A，and-863C/A，restriction enzymes（Sib Enzyme Ltd.，Novosibirsk，Russia）and products size are shown in Table 1.Primers specific for TNF-αgene sequences were synthesized by SibEnzyme（Novosibirsk，Russia）.