Quick Guide Extraction Deepwell Protocol Raw Beef Samples · • Prepare the PCR mix • Distribute...
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• Prepare the PCR mix • Distribute 20 μl/well in the PCR microplate • Start software • Create the plate setup • Start the amplification by clicking on “Run” probe mix amplification mix PCR mix 20 μl • Enrich the sample in pre-warmed SEB (375 g in 1,125 ml), 10-22 hrs at 41.5°C • Transfer 1 ml of the enriched sample in the deepwell plate Shake stomacher bag to homogenize, and allow to decant before collecting. Avoid collecting large fragments of food debris • Seal the deepwell plate with the plastic sealing film • Centrifuge at 2,250 g for 20 min • Discard all the supernatant (the DW 40 deepwell microplate washer can be used) • Add 200 μl of the complete lysis reagent (reagent A + F) Lysis reagent must be constantly stirring in order to keep it in suspension Use a micropipette with large opening 200 μl filter tips • Seal the deepwell plate with the pre-pierced sealing film 10-22 hrs 41.5°C SEB 200 μl 1 ml 20 min 2,250 g • Centrifuge at 2,250 g for 2 min 2 min 2,250 g • Incubate at 99°C for 15-20 min at 1,300 rpm in a plate agitator-incubator 15-20 min 1,300 rpm 99°C Please read the kit instruction manual and instrument user guide for complete and detailed instructions. • Add 5 μl of controls • Transfer 5 μl of sample supernatants Do not vortex before collecting the sample Check there are no bubbles • Seal the microplate 5 μl Quick Guide 357-8139 • iQ-Check ™ STEC VirX Standard II Extraction Deepwell Protocol Raw Beef Samples
Transcript of Quick Guide Extraction Deepwell Protocol Raw Beef Samples · • Prepare the PCR mix • Distribute...
• Prepare the PCR mix
• Distribute 20 μl/well in the PCR microplate
• Start software
• Create the plate setup
• Start the amplification by clicking on “Run”
probemix
amplificationmix
PCRmix
20 μl
• Enrich the sample in pre-warmed SEB (375 g in 1,125 ml), 10-22 hrsat 41.5°C
• Transfer 1 ml of the enriched sample in the deepwell plateShake stomacher bag to homogenize, and allow to decant beforecollecting. Avoid collecting large fragments of food debris
• Seal the deepwell plate with the plastic sealing film
• Centrifuge at 2,250 g for 20 min
• Discard all the supernatant (the DW 40 deepwell microplate washer canbe used)
• Add 200 μl of the complete lysis reagent (reagent A + F)Lysis reagent must be constantly stirring in order to keep it in suspensionUse a micropipette with large opening 200 μl filter tips
• Seal the deepwell plate with the pre-pierced sealing film
10-22 hrs
41.5°CSEB
200 μl
1 ml
20 min2,250 g
• Centrifuge at 2,250 g for 2 min2 min2,250 g
• Incubate at 99°C for 15-20 min at 1,300 rpm in a plate agitator-incubator
15-20 min1,300 rpm
99°C
Please read the kit instruction manual and instrument user guide for complete and detailed instructions.
• Add 5 μl of controls
• Transfer 5 μl of sample supernatants Do not vortex before collecting the sampleCheck there are no bubbles
• Seal the microplate
5 μl
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To find the correct volumes to use when preparing the PCR mix, add the total number of samples and controls tobe analyzed, and find the corresponding volumes of reagent B and reagent C in the table.
Quick Guide iQ-Check™ STEC VirXPCR Mix Calculation Guide
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000
Bio-RadLaboratories, Inc.
Life ScienceGroup
© Bio-Rad PF194 Rev B 06/2012
Total number ofsamples & controls
ProbesReagent B (μl)
Amplification MixReagent C (μl)
1 5 152 11 333 16 484 22 665 27 816 32 967 38 1158 43 1309 49 14710 54 16011 59 17712 65 19513 70 21014 76 23015 81 24516 86 26017 92 27518 97 29019 103 31020 108 32521 113 34022 119 35723 124 37024 130 39025 135 40526 140 42027 146 44028 151 45029 157 47030 162 48531 167 50032 173 52033 178 53534 184 55035 189 56536 194 58037 200 60038 205 61539 211 63540 216 65041 221 66542 227 68043 232 69644 238 71545 243 73046 248 74547 254 76048 259 777
Total number ofsamples & controls
ProbesReagent B (μl)
Amplification MixReagent C (μl)
49 265 79550 270 81051 275 82552 281 84553 286 86054 292 88055 297 89056 302 90557 308 92558 313 94059 319 96060 324 97061 329 99062 335 100063 340 102064 346 104065 351 105066 356 107067 362 109068 367 110069 373 112070 378 113071 383 115072 389 117073 394 118074 400 120075 405 121076 410 123077 416 125078 421 126079 427 128080 432 130081 437 131082 443 133083 448 134084 454 136085 459 138086 464 139087 470 141088 475 142089 481 144590 486 146091 491 147092 497 149093 502 151094 508 152095 513 154096 518 1550
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