Methods分析和总结分析和总结.docxVIP

Methods Select a fish – give it a unique number in each group will be numbered from 1-7 – keep track of your fish number. Weigh and measure the fish (standard length and fork length) – write this down Dissect out gastro-intestinal (GI) track – do not pierce the GI tract with the scalpel – be careful Use the pegs to ensure that no fluid escapes from the GI tract Label 1.5ml centrifuge tubes with your fish number Collect the fluid from the GI tract into labeled 1.5ml centrifuge tubes – keep cool in ice or in refrigerator Carefully rinse a section (1cm x 1cm) of upper GI tract with de-ionised water – collect in labeled 1.5ml centrifuge tube – store at -800C Collection of GI microbes Centrifuge collected GI fluid @ 7000 g for 10 minutes Take off supernatant – label and store at -800C – label 1F Resuspend pellet in PBS (1/2) Centrifuge resuspended pellet @3000 g for 3 minutes Keep supernatant (1/3) – discard pellet Centrifuge supernatant @ 3000 for 3 minutes Keep supernatant (1B) – discard pellet Centrifuge supernatant @ 10000 g for 10 minutes Keep pellet – store at -800C (1B) Sample preparation Microbial extract Thaw Add 175ul PPS to the microbes- vortex, xonicate, and centrifuge at 7000 g for 10 Mins Gut fluid Thaw Vortex and centrifuge at 7000g for 10 mins Amylase assay Samples and positive controls Add 50 ul samples, microbial extract and gut fluid, to 2 wells Amylase positive control-add 5ul tl 2 wells(duplicates),add 45ul H2O Standard curve 1) add 0,2,4,8,10ul of 2mM nitrophenol standard mix into 2 wells(duplicates),bring volume to 50ul with H2O Assay add 100ul reaction mix (1:1assay buffer : substrate mix) to each well mix and measure immediately at 405nm to obtain T0 incubate fo 30mins at 25℃H20 measure at 405nm o obtain T1 Calculation Plot the nitrophenol standard curve. Apply the absorbance T1-T0 to the nitrophenol standard curve to get B nmol of niteophenol generated by amylase between T0 and T1. Amylase activity =(B/(C*T)) * sample dilution factor = mmol/min/