Bacteria (tuf gene) Quantitative PCR Kit
Contents
1. Thermal Cycler Dice SYBR Molecular Probes Inc Premix Ex 7ag http wwwr takara bio coJp 14 RR240A http wwwr takara bio coJp 15 RR240A TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201406Da2. 1 SYBR Premix Ex Tag GC a SYBR Premix Ex 789 GC i 0 2 3 SYBR Premix Ex Tag GC SYBR Green 4 3
3. 1 g PCR 0 005 g 200 1 g 10 g 90 ml 1 ml 0 1 g DNA DNA 100 0 1 g 1 720 PCR PCR 5 u 0 005 g VI 2 1 q 1 copies g 2 1 9 copies g 1 1g 1 copies tube 2 1 copies tube 3 1 9
4. SYBR Premix Ex 7ag GC SYBR Green PCR GC 1 F F F n EE LL LL LL LI LLLLLIL 2 ux F er 3 1 DNA PCR DNA http www takara bio co jp 2 RR240A 25 100
5. 1 2 3 http wwwr takara bio coJp 5 RR240A V 100 ml mm 9 90 ml 109 eg DNA 1 ml 0 1g 8000 X g 5 1 1 100 DNA NucleoSpin Tissue DNA 100 ul PCR TUF Positive Control
6. PCR 4 PCR PCR PCR PCR 95 30 2 Step PCR 35 95 5 60 C 30 FAM SYBR PCR Tm http wwwr takara bio coJp 9 RR240A VI VI 1 TUF Positive Control TUF Positive Control tuf DNA OD26o 1 X 10 copies ul
7. Vy _ 0 1 9 5 ul 0 005 g PCR PCR 019 PCR http www takara bio co jp 6 RR240A V 1 2 10 9 90 ml 0 1 1 10g 2 9 90 ml 3 V 2 DNA 2 1 ml 8 000 Xg 5
8. 6 8 109 TSA 30 48 PCR 2 TSA 10 log copies g llog cuf g 151 090 s ah 3 wm 6 e 8 4 E e _ e PL 0 0 2 4 6 8 10 Tsaog chug 3 27 2 96 5 3 655 539 TSA CFU g PCR copieg TSA INR 0 1 2 a 2 RM EO http www takara bio co jp 11 RR240A VII Appendix 3 3 1 Food Prot 2010 Apr 73 4 670 9 4 10 ng DNA PCR
9. TUF Positive Control X 200 1 9 copies g http wwwr takara bio coJp 10 RR240A VI 3 Rahnella aguatilis Escherichia co7 Staphylococcus aureus TSB TSA 1 ml PCR 10 0 9 0 E 8 0 i 2 TSA CFU ml 9 ms PCR so 4 copies ml 4 0 1 R aquatilis TSA EE 0 4 La PCR 2 0 S aureus 1 0 0 0 0 2 0 4 0 6 0 8 0 10 0 TSA log CFU ml 2 9
10. DNA 5 PCR 1 3 TUF Positive Control EASY Dilution PCR 1 5 u 1 1 Xx 10 copies ul TUF Positive Control 2 1 Xx 104 copies ul TUF Positive Control 5 EASY Dilution 45 ub 3 1 Xx 103 copies ul 2 1 X 104 copies ul 5 ul EASY Dilution 45 ul 4 1 x 102 copies 1 3 1 X 103 copies ul 5 ul EASY Dilution 45 ul PCR copies g 1 9 VI 1 copies tube copies g copies tube copies g 1 19 2 1
11. o 20 ul 1 5 Thermal Cycler Dice Real Time System 1 SYBR Premix Ex 7gg GC 2 xX conc 12 5 ul TUF Primer Mix 5 X conc 5 0 wl dH20 25 ul 5 0 ul Total 25 0 ul Life Technologies PCR 1 SYBR Premix Ex Tag GC 2 xX conc 12 5 ul TUF Primer Mix 5 X conc 5 0 ul ROX Reference Dye or ROX Reference Dye ll 0 5 ul dH20 20 pl 5 0 ul Total 25 0 wl 1 StepOnePlus ROX Reference Dye 7500 Fast Real Time PCR System ROX Reference Dye I http wwwr takara bio coJp 8 RR240A 3 DNA 3 2 DNA 5 u 0 2 ml
12. Ct 20 Bacterial strains9 Strain Source Gram negative Bacteroidetes Chryseobacterium formosense FI55 Chub mackerel Flavobacterium hercynium FI48 Spotted mackerel Flavobacterium johnsoniae JCM 8514 Sejongia antarctica FI18 Spotted mackerel Sphingobacterium kitahiroshimence FI23 Stone flounder V Proteobacteria Acinetobacter baumannii JCM 6841 Acinetobacter baumannii Fl63 Flatfish meita Aeromonas hydrophila JCM 1027 Aeromonas molluscorum FI56 Horse mackerel Alteromonas macleodii JCM 20772 Citrobacter freundii IAM 12471 Colwellia aestuarii FI04 Chub mackerel Enterobacter aerogenes IAM 1183 Erwinia carotovora IAM 12633 Escherichia coli IAM 1137 Klebsiella pneumoniae IAM 1063 Morganella morganii ATCC 35200 Photobacterium phosphoreum FI59 Horse mackerel Proteus mirabilis JCM 1669 Pseudoalteromonas haloplanktis FIO1 Spotted mackerel Pseudomonas fluorescens FI28 Stone flounder Psychrobacter immobilis ATCC 43116 Psychromonas arctica FI26 Chub mackerel Rahnella aguatilis JCM 1683 Raoultella planticola JCM 7251 Salmonella serover Typhimurium ATCC 13311 Schineria larvae FI13 Spotted mackerel Serratia marcescens IAM 1104 Shewanella frigidimarina FI20 Spotted mackerel Shewanella japonica JCM 21433 Shewanella putrefaciens NBRC 3908 http www takara bio co jp 12 Accession number AB472770 AB472772 AB472806 AB472786 AB472789 AB4
13. NucleoSpin Tissue DNA 100 ul 1 1 ml 1 5 ml 2 8000 X9 5 3 20 mg ml lysozyme in 20 mM Tris HCL 2 mM EDTA 1 Triton X 100 pH8 0 1 180 ul 37 30 60 4 Proteinase K 2 25 uu 56 1 3 5 Buffer B3 200 ul 70 10 11 000 x 9 5 gt 99 210 ul NucleoSpin Tissue Column Collection Tube 6 11 000 Xx 9 1 Collection Tube 8 1 Buffer BW 500 11 000 X 9 1 C
14. 20 4 6 Package 2 20 http wwwr takara bio coJp 3 RR240A lt gt 0 1 lt DNA gt NucleoSpin Tissue 740952 10 50 250 gt 99 56 70 20 mg ml lysozyme in 20 mM Tris HCL 2 mM EDTA 1 Triton X 100 pH8 0 lt PCR gt PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Applied Biosystems 7500 Fast Real Time PCR System Life Technologies StepOnePlus Real Time PCR System Life Technologi
15. SYBR Premix Ex Tag GC 1 2 X conc 625 ul x 2 TUF Primer Mix 5 X conC 250 ul Xx 2 dH2O 1ml ROX Reference Dye 2 50 X conc 50 ul ROX Reference Dye II 2 50 X conc 50 ul TUF Positive Control 1 X 10 copies ul 100 wl EASY Dilution for Real Time PCR 1 ml X 2 1 TaKaRa Ex 7g9 HS dNTP Mixture Mg SYBR Green 2 Life Technologies PCR ROX Reference Dye 50 xX StepOnePlus Real Time PCR System Life Technologies ROX Reference Dye Il 50 Xx Applied Biosystems 7500 Real Time PCR System Life Technologies Applied Biosystems 7500 Fast RealTime PCR System Life Technologies Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 I 20 80 SYBR Premix Ex Tag GC Package 1 4 6 80 4 80
16. 72793 AB472767 AB472795 AB472768 AB472797 AB472801 AB472771 AB472802 AB472804 AB472805 AB472808 AB472816 AB472777 AB472817 AB472780 AB472781 AB472818 AB472783 AB472819 AB472820 AB472822 AB472788 AB472823 AB472787 AB472824 AB492873 tuf qPCR b amplification C 16 0 17 5 18 0 17 3 16 9 15 8 14 3 16 5 15 0 16 7 15 4 14 1 15 8 16 1 14 2 15 3 15 9 14 7 17 5 14 9 15 8 16 8 16 0 15 9 17 5 15 1 15 0 16 2 15 5 15 8 14 4 RR240A Vibrio diazotrophicus Vibrio parahaemolyticus Xanthomonas euvesicatoria Xanthomonas oryzae Yersinia enterocolitica a Proteobacteria Paracoccus denitrifcans amp Proteobacteria Alcaligenes faecalis Burkholderia caledonica Gram positives Bacilli Aerococcus sanguinicola Bacillus subtilis Brochothrix thermosphacta Carnobacterium divergens Carnobacterium maltaromaticum Enterococcus faecalis Lactococcus lactis Leuconostoc carnosum Listeria monocytogenes Planomicrobium chinense Staphylococcus aureus Staphylococcus pasteuri Actinobacteria Rothia dentocariosa Rothia nasimurium Salinibacterium amurskyense FI52 ATCC 17802 FI22 JCM 20241 ATCC 9610 FI34 JCM 20522 JCM 21561 JCM 11549 IAM 1076 NBRC 12167 JCM 5816 NBRC 15684 JCM 5803 NRIC 1174 JCM 9695 ATCC 15113 FI41 ATCC 12600 FI64 JCM 3067 FI43 FI36 Horse mackerel Chub mackerel Spotted mackerel Chub mac
17. es http www takara bio co jp 4 RR240A IV 3 2 3 121 20 2 59 0
18. fect Real Time RR071A B Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 NucleoSpin Tissue 740952 10 50 250 Enterobacteriaceae p P gene Quantitative PCR Kit RR241A IX 1 118 71 79 1989 2 35 20 JournalofNational Fisheries University 60 4 183 188 2012 3 141 6 4 Tanaka Y Takahashi H Simidu U Kimura B Design of a new universal rea time PCR system targeting the tuf gene for the enumeration of bacterial counts in food Food Prot 2010 Apr 73 4 670 679
19. kerel Flatfish meita Horse mackerel Flatfish meita AB472791 AB472827 AB472792 AB472828 AB472829 AB472779 AB472796 AB472799 AB472794 AB472798 AB492875 AB472800 AB492874 AB472803 AB472811 AB472812 AB472813 AB472778 AB472826 AB472790 AB472821 AB472784 AB472785 16 2 16 3 15 5 19 6 15 9 17 0 17 9 16 3 18 4 16 8 15 3 15 7 16 2 14 8 18 1 17 9 18 4 16 0 16 1 16 0 16 1 18 2 17 2 a Classificaiton was based on the National Center for Biochnology Information taxonomy b The cycle threhold value Ct for the reaction containing 10 ng of genomic DNA per reaction zu GenBank 1 561 2011 11 21 tuf elongation factor 10 855 100 http wwwr takara bio cojJp cycleave elongation_factor xlsx http wwwr takara bio coJp 13 RR240A VII SYBR Premix Ex Tag GC Per
20. mma RR240A IdfaR4 Bacteria tuf gene Quantitative PCR Kit vV201406Da PCR 2 4 3 PCR 16S rRNA 16S rRNA 1 2 Tu tu PCR PCR PCR
21. ollection Tube 2 Buffer B5 3 600 11 000 X 9 1 Collection Tube 9 11 000 X g 1 10 1 5 ml 70 Buffer BE 100 ul 1 11 000 x g 1 DNA 4 20 1 NucleoSpin Tissue 2 Proteinase K 740952 10 Proteinase K 6 mg Proteinase Buffer PB 260 ul Proteinase K 20 6 3 Buffer B5 740952 10 Wash Buffer B5 concentrate 4 ml 96 100 16 ml http wwwr takara bio coJp 7 RR240A V 3 PCR V 2
Download Pdf Manuals
Related Search