Fungi-Plex Kit HOW TO USE IT F E C C. Wash Buffer [F] Standard Diluent Buffer [E] Assay Diluent Buffer [D] Mycotoxin Standard Mix

Transcription

1 TM ungi-plex Kit W T U IT tandard iluent uffer [] Mycotoxin tandard Mix ssay iluent uffer [] etection Reagent ead Mix t i K x Wash uffer [] TY I T u U e h T I I I V Y PR I Y R T Y M T Y W T I T R 9 9 P P Y U R V I I R U R e l P ngi

2 ungi-plex Kit TP. ow to use it ollow the quantity guide tables next page. dil. Wash uffer [] ssay iluent uffer [] distilled water UTPUT abel the results by [diluted ] IPUT dil. ungi-plex kit includes three buffers, out of which, two should be diluted. Wash uffer [] and ssay iluent uffer [] are in 0X concentrated. These buffers should be diluted by distilled water. UTPUT 00 abel the results by [diluted ] acetonitrile distilled water vortex shaker Mycotoxin tandard Mix IPUT shaking: 30 sec conditioning: 0 min 00 The standard mix vial [] contains lyophilized mycotoxins. The mix must be dissolved in 00 acetonitril (app. 00%)and after a few minutes (0 min), after this add further 00 distilled water, mix it. eave the reconstituted mix to equilibrate for at least 0 minutes before starting the preparation of standards. abel the results by [rec. ]. vortex shaker rec. 50X UTPUT shaking: 30 sec conditioning: 0 min

3 ilution guide (quantity guide tables for TP.) umber of samples Wash uffer [] - quantities to dilute Wash uffer [] istilled water / d 77,9 7 00,, ,5 53,0 3 7,0 39,0 5,0 090,0 36 0,0 5 7,0 5 09,0 7 6, ,0 65, 73 6, Total 7 79,0 5,0 5 30,0 3 90, ,0 57 0,0 7 60,0 65,0 umber of samples ssay iluent uffer [] - quantities to dilute istilled water / d ssay iluent uffer [] 3,5 75,0,3 793, 3, ,0 633,5 5 70,5,5 0 9,5 79,5 6 3,5 370,5 33,5 60, 3,9 Total 3,5,5 3 0, ,0 5,0 7 95, ,0 6 0,0

4 l ground sample Whatman o. 595 / filter 5 m 5g TP. orbital shaker % acetonitrile* IPUT 0 min ach sample that is measured should weigh at least 5g. or extraction weigh 5g of the ground sample, add 5 ml of % (v/v) acetonitrle/d. hake it with an orbital shaker for 0 minutes. ilter the extract through a filter (e.g. Whatman o. 595 / filter). 0 0 diluted filtered sample diluted sample 5x dil. dil. UTPUT IPUT ilute the filtered sample by adding 0 of ssay iluent uffer [diluted ] to 0 of the sample. [diluted sample 5x] distilled water 00% acetonitrile IPUT m l 6 ml * ow to create % acetonitrile % acetonitrile UTPUT

5 TP. xample for sample ollow the quantity guide at bottom of the page. ead preparation ead Mix 539 abel the result by [diluted ]. dil. dil. dil. IPUT UTPUT ach tube or well requires 50 of diluted bead mix. The mix has to be made by adding of ead Mix [] to 9 of Wash uffer [diluted ]. In each case 00 of additional diluted mix should be used, because there is a certain amount of waste in every measurement. 5 sec 5 sec Vortex the ead Mix [] for 5 seconds, and sonicate for 5 seconds. umber of samples ample tandars Waste Wash uffer Wash uffer Wash uffer ead Mix [] ead Mix [] ead Mix [] [dil. ] [dil. ] [dil. ] Total ead Mix [] Wash uffer [dil. ]

6 tandard iluent uffer [] 90 0 Preparation of standards TP. 50x : rec. 50X IPUT rec. 50X abel seven ppendorf tubes and arrange them in the following order: 50x (:), :, :, :, :6, :3 and : ilute the Mycotoxin tandard Mix [rec ] solution in tube '50x' by adding 90 of tandard iluent uffer [] to 0 Mycotoxin tandard Mix [rec ]. 50x : : : : :6 : : :6 :6 500 dd 500 of tandard iluent uffer [] to each of the remaining tubes. 50x : x : 500 : 500 : UTPUT Perform a serial dilution by transferring 500 from the 50x (:) (Top tandard) to the : dilution tube and mix thoroughly. ontinue making serial dilutions by transferring 500 from the : tube to the : tube and so on to the :6 tube and mix thoroughly. It is recommended that the first eight wells or tubes in the experiment should be the standards. tandards to be run in order from least concentrated (tandard iluent uffer, 0 ng/ml) to most concentrated (:). tandard zero is the tandard iluent uffer []. :3 :6

7 TP 3 xample for sample. etection Reagent dil. IPUT otification: detection reagent is light sensitive, use tinfoil, dark tubes or other darkening solutions. umber of samples ,5 55,5 µ l Preparation of the detection regent dil. R mix x ach tube or well requires 50 of diluted etection Reagent. The mix has to be created by adding 0,5 of etection Reagent [] to 9,5 of ssay iluent uffer [diluted ]. In each case 00 of additional diluted mix should be used, because there is a certain amount of waste in every measurement. UTPUT abel the tube by [R Mix x] and store it at. ample ample Waste Total ssay iluent ssay iluent ssay iluent ssay iluent etection etection etection etection uffer uffer uffer uffer Reagent [] Reagent [] Reagent [] Reagent [] [dil. ] [dil. ] [dil. ] [dil. ] 0,5 9,5 5,5 5,5,5 7,5 7,5 7,

8 TP / tandard iluent uffer [] (zero standard) ssay procedure on filter plates diluted sample 5x dil. R mix x : : : :6 :3 : x : 00 IPUT IPUT dd 00 of the zero standard, also add 00 of the standards (labelled :6 50x, :3, :6, :, :, :, and (:),) to the wells. tandards should be run in order from least concentrated (tandard iluent uffer [], 0 ng/m) to most concentrated (:). :6 :3 :6 : : : 50x :

9 TP / ssay procedure on filter plates dd 00 of the samples to the wells. [sample x] 00 sample x 50 dd 50 of the diluted etection Reagent mixture [R Mix x] to each well. (Protect from direct exposure to light as much as possible.) R mix x dil. 50 Vortex the diluted ead Mix [] for 5 seconds. dil. 5 sec dd 50 ead Mix [diluted ] to each well.

10 TP /3 ssay procedure on filter plates otification: Wells now are light sensitive, use tinfoil or other darkening solutions. Incubate the microwell plate for 5 minutes, using a /digital/ shaker at 600 RPM. orbital shaker wells after incubation UTPUT 5 min 600 RPM

11 TP 5 Insert the plate to the vacuum manifold and aspirate (do not exceed the 0 kpa!) until the wells are totally drained (-0 seconds). Washing procedures vacuum manifold wells after incubation dil. IPUT -0 sec dd 00 of the Wash uffer [diluted ] to each well. spirate again (do not exceed the 0 kpa!) until the wells are totally drained (-0 seconds). 00 Repeat the washing (previous) step two times. 3x dil. -0 sec dd 00 of the Wash uffer [diluted ] to each well. hake the microwell plate on a /digital/ shaker at 600 RPM for 5 minutes to resuspend the beads or mix it with your pipette. 00 Wipe the bottom of the plates! dil. orbital shaker 5 min 600 RPM UTPUT wells are ready to measure and analyse

12 TP 6 nalyse If you perceive any of the following signs, check your device and the procedure. - The number of beads/wells are far below The cytometer analyses a well for more than 3 minutes. or the analysis use P rray software. creens from the software: