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DNA Protocol

1. cecal oe 10 1 10 50 star
2. Cap Install plug click 3 Sample tray separation buffer Tag Access plate click Start click GO Sample tray Buffer Tray Plate select Right Plate plate Loaded Immerse Capillaries check Load or Cancel click A CEQ8800 Main switch OFF 31 5 PCR W 1 PCR 2 Ligation TA c1oning 1 3 invitrogen TOPO TA cloning kit for sequencing reagent PCR Gns
3. DNA 11 2 4 Purification of PCR products by PEG precipitation PEG polyethylene glycol 8000 13 PEG 1 6M NaCl autoclave 1 PCR products 20u 1 PEG solution 20ul on ice 30 min 2 centrifuge 12 000rpm 4C 20min 3 NA 4 rinse 70 EtOH 1001 centrifuge 12 000rpm 4C 5 min 5 dry 6 7 1 2 6 15 25p 1 DW 7 1u 1 DNA 1 agarose gel L DNA 12 2 5 restriction enzyme introduction restriction enzyme restriction endonuclease DNA
4. Plate Right Plate plate Loaded O Immerse Capillaries check Load click Sample Data Main menu icon SET UP click create a new sample plate OK 1 Sample sheet data 2 Cell lane Method LFR 1 3 Cell well lt Tab gt Analysis click Perform Analysis check LT Parameter Set default Sequence Analysis Parameter select 4 Tool bar File BELG save as project name Fail DK click 5 Run sample plates click Save to project project project default folder Plate Load click 6 OK 30 He at 1 Run control click Tab Direct Control click 2 Dummy cartridge Tab Life click Remaining Icon gel cartridge click gt OK click GO
5. 150 200bp 200bp 34 6 Basic Reagents CHAOS solution not autoclave Reagent Amount Final Conc Guanidine thiocyanate 50g 4M Salkosyl 0 5g 0 5 Tris HCl pH 8 0 1M stock 2 5 ml 25ml 2 Mercaptoethanol 0 7 ml 0 1M dH20O Up to 100 ml Salkosyl N lauroy sarcoseine sodium N Dodecanoyl N methylglycine sodium 2X CTAB solution not autoclave Reagent Amount Final Conc EDTA pH 8 0 0 5M stock 2 ml 20mM Tris HCl pH 8 0 1M stock 5 ml 100mM NaCl 5M stock 14 ml 1 4M CTAB 2g 4 dH2O Up to 50 ml CTAB EDTA Tris NaCl dHL0 stock oF i RE gt ASY DNA extraction buffer autoclave Reagent Amount Final Conc EDTA pH 8 0 0 5M stock 5 ml 50mM Tris HCl pH 8 0 1M stock 2 5 ml 50mM NaCl 2 34 g 0 4M dHzO Up to 100 ml 0 5M EDTA pH 8 0 autoclave Reagent Amount Final Conc EDTA 186 1 g 1M dH20 Up to 100 ml EDTA 2Na 2H 0 MW 372 24 EDTA 4Na Na
6. NE NT 37 Taq I 65C C U 10 50 1 37 C 1 1 g ADNA 13 2 6 Identification of Symbiodinium clade 2 6 1 Digestion of 18S rRNA with restriction enzyme identification of Symbiodinium clade DW Taq 1 Taq polymera Symbiodinium Taq I 1 DNA se PCR Taq 1 PCR Reagent Amount Final Conc
7. DS CTAB DNA Coffroth et al 1992 CTAB solution 20 mM EDTA 100 mM Tris HCl pH 8 0 4M NaCl 2 CTAB 0 2 2 mercaptoetanol add Proteinase K final concentration 0 1 mg ml 2X CTAB solution Hirose original CHAOS solution DNA extraction buffer CTAB solution Reagent Amount Final Conc EDTA pH 8 0 0 5M stock 2 ml 20 mM Tris HCl pH 8 0 1M stock 5 ml 100 mM NaCl 5M stock 14 ml 14M CTAB 2g 4 dH20 Up to 50 ml CTAB stock I CHAOS CTAB solution CHAOS solution 3001 2X CTAB 300 u 1 65 C 30 60min EDTA Tris NaCl DW 1 1 DNA extraction CHAOS methods DNA II DNA extraction buffer CTAB solution
8. 8 000rpm 1min 4 C 5 Buffer AW1 500n1l 8 000rpm 1min 4 C 6 Buffer AW2 500ul 8 000rpm 3min 4 C 7 DNeasy Mini spin colum 1 5ml kit Buffer AE 100u1 3 re 8 8 000rpm 3min 4 C 1 9 Sul Nano drop lul 24un1l PCR mixture AquaPure Genomic DNA Isolation Kit BIO RAD CHAOS solution DNA Hi 0 CHAOS 1 CHAOS solution 100ul Lysis buffer DNA extraction buffer 200ul 55 C 1h 2 Protein Precipitation Solution Kit 100ul voltex 10min ZOE 12 000rpm 5min 4 C 3 1 5ml 350 400401 1006 12 000rpm 15min 4 C 4
9. Reagent Amount Final Conc PCR 5 ul 2 10X Taq I buffer 1 0 ul 1X 3 0 1 BSA 1 0 ul 0 01 4 Taq I 10U n 0 2 ul 1 dH 0 2 8 ul 2 PCR 65C 3 3 4 4 agarose gel Syner gel 1 65 0 165g 10m1 agarose 0 7 0 07g 10m1 3 1 TAE 100 ethanol 2ml 1 sg Syner gel agarose 3 2 4 DNA 1oading buffer 100bp 16 Taq I ha 1 28S rDNA Symbiodinium Taq A B C C D F Hha 17 3 PCR DGGE profiling of Symbiodinium types using ITS2 r
10. 3M NaOAC pH 5 2 1un1 sample 100mM EDTA pH 8 0 lul sample 20mg ml Glycogen 0 5u1 sample 3 2 sequencing reaction stop solution 2 5n1l 100 EtOH 30nl 3 3 centrifuge 14 500rpm 4C 15 min 3 4 NA Fa TOPRCBRBUCWSR 3 5 rinse 70 EtOH 125 1 centrifuge 12 000rpm 4C 3min 3 6 DNA Fa TOPRCBRBUCWHSR 27 3 7 rinse 2 70 EtOH 125 1 centrifuge 12 000rpm 4C 3min 3 8 3 9 dry 6 7 1 2 3 10 30u1 SLS 4 C 1 20C 1 3 11 96 PCR 3 12 4 separation buffer 96 80 28 4 2 CEQ 8800 l PC main switch ON Ctr Alt Del Pass word gt
11. 3 DNA Il DNA 1 2 QIAGEN DNA DNA 1 2a DNA isolation by standard phenol chloroform method 1 CHAOS solution 300 u 1 amp 1 5ml 7 OF a TICKL incubate 55 C 1h 2 Tissue CHAOS solution 3001 Phenol TE 150 1 CTA 150 u 1 mix by voltex centrifuge 14000rpm 4 C 15min lt 2 3times gt depend on specimen Phenol TE TE pH 8 0 CIA 24 1 3 Aqueous phase 300u 1 CIA 300 1 mix by voltex centrifuge 14000rpm 4 C 15min 4 Aqueous phase 300 uw 1 3M NaOAC pH 5 2 3041 1 10 Isoropanol 30041 gently mix incubate 20C 30min centrifuge
12. 94 C Cycle 94 C 3 00 min 1 cycle J 94 C 1 00 min 58 C 1 00 min 35 cycles 72 C 2 30 min J 7 00 1 cycle J 4 C hold 3 PCR products 51 1 agarose gel Small subunit ribosomal RNA primers ss5Z ss3Z K1 500 1600bp 2 2 PCR amplification of large subunit ribosomal RNA 28S zoox specific primer Zadoya et al 1995 lsu UFP1 5 CCC GCT AAT TTA AGC ATA TAA GTA 3 lsu URP1 5 GTT AGA CTC CTT GGT CGT GTT TCA 3 1 PCR Reagent For 25u PCR For 50ul PCR Template DNA Xu1 8 25ng Xu 1 5 50ng 2 10 X PCR buffer 2 5 ul 5 ul 3 dNTP 2 5mM each 2 ul 4 ul 4 primerl 10 pmol 0 5 ul 1 ul 5 primer2 10 pmol 0 5 pl l1 ul 6 Taq polymerase 5U u 1 0 125 ul 0 25 ul 1 dH 0 Up to 25 ul Up to 50 ul 1 1 FU PVRS OREReEMNOBFIAICL SOV ZaFa TILANT premix 1 2 24 1 49 1 0 2m1l template DNA 2 94 Cycle 94 C 5 0
13. return CEQ 8800 main switch ON 2 icon CEQ click 3 Icon Run click Error message DK click Tab Direct Control click 4 Gel cartridge gel cartridge icon click DONE Cap Lot Number Hours on Instrument DONE click Tab Life click Remaining 1Run 0 8ml 5 Sample tray separation buffer Tag Access plate click Start click GO Wetting tray DW change Sample tray well Al 29 Buffer tray
14. PCR GC clump ITS2 ITSintfor2 ITSUP l i 18S ITS1 5 88 ITS2 28S lt ITSrev2 X rDNA 5 ITS PCR zoox specific primer ITSUP Santos et al 2001 ITSrev2 LaJeunesse and Trench 2000 ITSUP 5 CCG GTG AAT TAT TCG GAC TGA CGC AGT GCT 3 ITSrev2 5 GGG ATC CAT ATG CTT AAG TTC AGC GGG GT 3 5 1 PCR Reagent For 25ul PCR Template DNA Xu 1 3 25 ng 2 10 X PCR buffer 2 5 ul 3 dNTP 2 5mM each 2 ul 4 primer 1 10 pmol 0 5 ul 5 primer 2 10 pmol 0 5 ul 6 Taq polymerase 5U 1 0 125 pl 1 dH 0 Up to 25 ul 19 5 2 94 Cycle 94 C 5 00 min 1 cycle J 94 C 0 30 min 55 C 0 30 min 30 cycles 72 C 1 30 min J 72 C 10 00 1 cycle J 4 C hold 6 GC clump ITS2 PCR TITSintfor2 ITSrev2GCC touch down ITS2 PCR Reagent For 50u PCR PCR products 0 5 ul 2 10 X PCR buffer 5 ul 3 dNTP 2 5mM each 4 ul 4 primer1l 10 pmol 1 ul 5 primer2 10 pmol 1 ul 6 Taq polymerase 5U u 1 0 25 ul 1 dH 0 36 25 ul 20 3 2 P
15. PCR 50 100 ng 2 10X Taq I buffer 1 0 ul 1X 3 0 1 BSA 1 0 ul 0 01 4 Taq 1 10U nu1 0 2 ul 1 dH 0 Up to 10 ul PCR 7ag 1 Reagent Amount Final Conc PCR 5 ul 2 10X Taq I buffer 1 0 ul 1X 3 0 1 BSA 1 0 ul 0 01 4 Taq 1 10U nu1 0 2 ul 1 dH 0 2 8 ul PCR 0 2 ml premix 2 3 2 5 Syner gel 0 9 agarose 0 7 1 SMI OV 4 PCR 65C 3 agarose gel 0 09g 10m1 0 07g 10m1 3 1 agarose TAE Sora CLS Z El 14 UL CHE ZEB DRETA R 4 DN
16. mercaptoethano guanidine salt chaotropic agents rc RAE CHAOS solution 1 CHAOS solution 2m1 7m1 DRA A 1 2 OK 2cm 2cm A 2 5cm
17. 15 touch down 18 heteroduplex 3 PCR products 51 1 agarose gel ITS2 ITSintfor2 ITSrev2GC 300bp 4 PCR 2 3 2 4 PCR 45 1 12 1 DW 1 1 DGGcE 5x 1 genomic DNA PCR ITS2 genomic DNA PCR PCR ITS ITS1 5 8S ITS2
18. lt gt L 70 EtOH 209C RNase 2 DNA extraction buffer 500 u 1 1 3 DNA extraction buffer 300 14 4 3001 2X CTAB solution 300 1 10 SDS 60 1 2 melcaptethanol 13 1 5 6 total 700 1 extraction CHAOS methods RNase 65 C lh DNAX HH PC 10mg ml Proteinase K 33 u1 37 C BE 3 5 1 1 DNA gt 1 1d Others 1 DNA 1 Water pick DNA extraction buffer 2
19. 12 DGGE OH DNA 6 EtBr 30 UV 7 I PCR 1 blue 1 5m1l 2 lt TE 20nl 4 Il DGGE PCR 1 PCR dHO 100n1 2 blue PR 3 dH O PCR 25ul PCR PCR B lt 8 DNA Zoox specific ITS2 primer LaJeunesse and Trench 2000 ITSin
20. 100 denaturing soln 9 6 ml 4 8 ml DCode Dye soln 320 ul total 16 ml a 16 ml 3 3 high solution 3 3 1 high solution 16ml TEMD 14 4ul 10 3 3 2 10 APS 144nul 10 3 3 3 IRZ 3 4 low solution 3 3 3 5 3 5 1 3 5 2 Y 5 3 5 3 3 6
21. 2 1 2a DNA isolation by standard phenol chloroform method 1 2b DNA DNA D isolation by commercial kit 1 1b SDS Proteinase K method CHA0S methods DNA I NA 5 15m1l RNase DNA extraction buffer SDS Proteinase K pce sla ear es eels casa Wana DNA extraction buffer 1 1c CTAB method DNA extraction
22. 70 800ml 12 000rpm 5min 49C 5 30 60nin 15min 6 DNA Hybridization Solution Kit 50ul DNA 1 9 Bul Nano drop lul 24unl PCR mixture 2 PCR RFLP identification of Symbiodinium clade 2 1 PCR amplification of small subunit ribosomal RNA 18S rDNA zoox specific primer Rowan et al 1991 ss5Z 5 GCA GIT ATA A G TT TAT TTG ATG GT C T A G CT GCT AC 3 ss3Z 5 AGC ACT GCG TCA GTC CGA ATA ATT CAC CGG 3 1 PCR Reagent For 25u PCR For 50u PCR Template DNA Xu 1 3 25ng Xu 1 5 50ng 2 10 X PCR buffer 2 5 ul 5 ul 3 dNTP 2 5mM each 2 ul 4 ul 4 primerl 10pmol 0 5 ul 1 ul 5 primer2 10pmol 0 5 ul 1 ul 6 Taq polymerase 5U u 1 0 125 ul 0 25 ul 1 dH 0 Up to 25 ul Up to 50 ul 1 1 1 5ml premix 1 2 24 1 49 1 0 2ml template DNA 2
23. 0 min 1 cycle J 94 C 0 45 min 52 C 0 45 min 35 cycles 72 C 1 30 min 72 C 7 00 1 cycle J 4 C hold 3 PCR products 5u 1 amp 1 agarose gel Large subunit ribosomal RNA primers lsu UFP1 1su URP1 W 700bp 10 2 3 Purification of PCR products by ethanol precipitation DNA 1 PCR products 20 1 45 1 3M NaOAC pH 5 2 2ul 4 5u1 1 10 volume isopropanol 25 1 50u1 approx equal volume 1 20 C 30min 20 C DNA 2 centrifuge 12 000rpm 4C 25min 3 DNA HOCH 4 rinse 70 EtOH 18041 centrifuge 12 000rpm 4C 5min 6 dry 6 7 1 2 7 15 251 DW PCR 25un1 PCR 15ul DW 8 1 1 DNA 1 agarose gel L
24. 14000rpm 4 C 25min 5 Aqueous phase waste 70 EtOH 500 ul centrifuge 14000 rpm 4 C 5min 6 Dry T resuspend in 50 100u1 TE lt Option gt 8 RNase A 10mg ml lul Incubate 37 C 1h 9 add TE up to 100u 1 Phenol TE 50ul CIA 50 1 mix by voltex centrifuge 14000rpm 4 C 20min 10 Aqueous phase 100 1 CIA 100 1 mix by voltex centrifuge 14000rpm 4 C 20min 11 Aqueous phase waste 100 1 3M NaOAC pH 5 2 10p 1 Tsopropano1 100 1 gently mix incubate 20 C 30min centrifuge 14000rpm 4 C 25min 12 Aqueous phase waste 70 EtOH 300 u 1 centrifuge 14000rpm 4 C 5min 13 Dry resuspend in 30 50u1 TE 1 agarose gel 1 51 1 2b DNA isolation by commercial kit DNeasy Blood amp Tissue Kit QIAGEN CHAOS solution DNA FH 0 CHA0S solution 1 CHAOS solution 100ul Buffer ATL DNA extraction buffer 100ul voltex THA LE 1 an h 55 C lh 2 voltex 20 PIR LER Buffer AL 200ul voltex CHEL 70 C 10min 3 100 EtOH 200n1 voltex DNA 4 DNeasy Mini spin column 2ml collection tube
25. 2008 3 27 Mamiko HIROSE Protocols for Symbiodinium identification ver 2 0 Table of contents 1 DNA extraction l la CHAOS method _ 2 1 1b SDS Proteinase K method _ 2 1 1c CTAB method 4 1 1d Others Frozen sample Ethanol fixed sample 5 1 2a DNA isolation by standard phenol chloroform method _ 6 1 2b DNA isolation by commercial kit _ 7 2 PCR RFLP identification of Symbiodinium clade 2 1 PCR amplification of small subunit ribosomal RNA 18S _ 9 2 2 PCR amplification of large subunit ribosomal RNA 28S _ 10 2 3 Purification of PCR products by ethanol precipitation 11 2 4 Purification of PCR products by PEG precipitation 12 2 5 Restriction enzyme introduction 13 2 6 Identification of Symbiodinium clade 2 6 1 Digestion of 18S rRNA with restriction enzyme 14 2 6 2 Digestion of 28S rRNA with restriction enzyme 16 3 PCR DGGE profiling of Symbiodinium types using ITS2 region 3 1 PCR amplification of ITS2 with GC clump _ 18 3 2 Preparation of gradient gel 21 4 S
26. 7L 1X TAE buffer buffer 2 3 buffer 5 10 4 1 1X TAE buffer 4 2 24 4 3 FEARED 7b eekly bh 4 4 PCR 25 5l 2X Loading Dye 4 5 DNA 38ul 2X Loading Dye3 ul loading buffer 35 50 DGGE 2X Loading Dye 70 5 60 CO 100V
27. A 1oading buffer 100bp Clade 1oading buffer 0 2 BPB Bromo pheno blue 2mM EDTA pH 8 0 50 glycerol in Mi11iQ 1oading buffer X6 X10 1oading buffer 1oading buffer 1000 bp 700 bp 500 bp 400 bp 300 bp 200 bp 100 bp Fig 2 n18S rDNA Taql generated RFLPs 1 clade A 2 clade B 3 clade C 4 clade D 5 clade D 6 clade D 7 clade E 8 from P damicornis Santos et al 2000 MPE 23 97 111 15 2 6 2 Digestion of 28S rRNA with restriction enzyme identification of Symbiodinium clade Taq I 1 Reagent Amount Final Conc PCR 50 100 ng 2 10X Taq I buffer 1 0 ul 1X 3 0 1 BSA 1 0 ul 0 01 4 Taq 1 10U nu1 0 2 ul 1 dH 0 Up to 10 ul k PCR 7ag2 1
28. OH EDTA pH pH 5N NaOH NaOH ryt 35 5M NaCl autoclave Reagent Amount Final Conc NaCl 14 6 g 5M dH20O Up to 50 ml 13 PEG solution autoclave Reagent Amount Final Conc PEG 8000 6 5 g 13 NaCl 4 67 g 1 6M dH20 Up to 50 ml 10 SDS not autoclave Reagent Amount Final Conc SDS 5g 10 dH20 Up to 50 ml 3M NaOAc sodium acetate Reagent Amount Final Conc NaOAc 3H20 20 4 g 3M dHzO Up to 50 ml pH pH pH5 2 eT ul k 50 x TAE autoclave Reagent Amount Final Conc Tris 242 g 2M Acetic acid 57 1 ml 2M EDTA pH 8 0 Stock 0 5M 100 ml 0 05 M dH20 Up to IL 36 5 x TBE autoclave Reagent Amount Final Conc Tris 54 g 0 445M Boric acid 27 5 g 0 445M EDTA pH 8 0 Stock 0 5M 20 ml 0 01 M dH20 Up to IL 10 x TE autoclave Reagent Amount Final Conc Tirs
29. Reagent Amount Final Conc Bromophenol Blue 0 05g 0 5 Xylene Cyanol 0 05 g 0 5 1X TAE buffer 10 0 ml 1X 21 c 2X Gel Loading Solution Reagent Amount Final concentration 2 Bromophenol Blue 0 25 ml 0 05 2 Xylene Cyanol 0 25 ml 0 05 100 Glycerol 7 0 ml 70 dH2O 2 5 ml Total volume 10 0 ml d DNA marker mp KEER L OTEA DNA 1 1 5 2ug DNA DNA 1 Reagent Amount Final Conc DNA 0 3ug ul 200 ul 60 ug 10X 72 1 buffer 40 ul 1X 0 1 BSA 40 ul 0 01 Taq I 10U ul 16 wl dH20 104 ul 100 u 0 2mlPCR 2 65C 3 95C 10 3 120 nl TE 0 5ng u1 1 3n1 1 5ung e 10 APS Ammonium Persulfat
30. buffer autoclave DNA Reagent Amount Final Conc EDTA pH 8 0 0 5M stock 5 ml 50 mM Tris HCl pH 8 0 1M stock 2 5 ml 50 mM NaCl 2 34 g 0 4 M dH20 Up to 100 ml 10 SDS not autoclave 10mg ml Proteinase K not autoclave store at 20 C 1 70 100 EtOH DNA extraction buffer M111i Q 20 C O 2 SDS final 1 2 fina1 2 657C 1h Rn 3 Proteinase K final 0 5 mg ml 379C 4 RNase E 1 2a DNA isolation by standard phenol chloroform method 1 1c CTAB method CTAB Cetyltrimethylammnonium bromide CTAB
31. e Reagent Amount For one plate For two plates Ammonium Persulfate dH20 0 5 g 500 ul 0 8 g 800 ul 22 f 0 Denaturing solution Reagent Amount Final Conc One plate Two plates 40 Acrylamide Bis 4 ml 7 4 ml 8 50X TAE buffer 400 pl 740 ul 1X dH20 up to 20 ml 37 ml g 100 Denaturing solution Reagent Amount Final Conc One plate Two plates 40 Acrylamide Bis 3 2 ml 6 ml 8 50X TAE buffer 320 ul 600 ul 1X Formaide 6 4 ml 12 ml 40 Urea 6 72 g 13 44 g 7M dHzO up to 16 ml 37 ml Urea 2 100 MilliQ 96 XD PCR 4 3 23 3 2 high solution low solution 0 100 solution 50ml High soln Low soln 0 denaturing soln 6 4 ml 11 2 ml
32. egion 3 1 PCR amplification of ITS2 with GC clump zoox specific ITS2 primer LaJeunesse and Trench 2000 ITSintfor2 5 GAA TTG CAG AAC TCC GTG 3 ITSrev2GC 5 CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GGG ATC CAT ATG CTT AAG TTC AGC GGG GT 3 GC clump 1 PCR Reagent For 25ul PCR For 50ul PCR Template DNA Xu 1 3 25 ng Xu1 5 50 ng 2 10 X PCR buffer 2 5 ul 5 ul 3 dNTP 2 5mM each 2 ul 4 ul 4 primerl 10 pmol 0 5 ul 1 ul 5 primer2 10 pmol 0 5 ul 1 ul 6 Taq polymerase 5U u 1 0 125 ul 0 25 ul 1 dHLO Up to 25 ul Up to 50 ul 1 1 PY PVE ORES MN OF SIBIC 41 5ml T premix 1 2 241 49p 1 0 2 mm template DNAX 2 94C Cycle touch down methods 94 C 5 00 min 1 cycle J 94 C 0 45 min KC 0 45 min 20 cycles 72 C 1 00 min J 94 C 0 45 min 52 C 0 45 min 15 cycles 72 C 1 00 min J 72 C 20 00 1 cycle J 4 C hold touch down methods 62C 1 0 5 9C 5290C 52C
33. equencing 4 1 Sequencing reaction for Beckman CEQ 8800 27 4 2 CEQ 8800 operation 29 5 PCR products sub cloning 31 6 Basic Reagent 35 1 DNA extraction 1 1a CHAOS method genomic DNA PCR DNA CTAB solution DNA 1 1c CTAB methods CHAOS solution Reagent Amount Final Conc Guanidine thiocyanate 50 g 4M Salkosyl 0 5 g 0 5 Tris HCl pH 8 0 1M stock 2 5 ml 25ml 2 Mercaptoethanol 0 7 ml 0 1M dH20O Up to 100 ml guanidine mercaptoethano1l 3 p mercaptoethanol CHAOS solution mercaptoethanol 7u1 m1
34. ert M Amount Vector pCR 2 1 3956bp 10ng nul Salt solution DNA dH 0 5 30 3 LB 3 1 LB 1L l ul l ul 11 7 ng 100bp lt 4n1 Up to 6 ul reagent Amount Final Conc Bacto tryptone 10 g 1 Bacto yeast extract 5 g 0 5 NaCl 10 g 1 5N NaOH 0 2 ml Bacto agar 15 g 1 5 3 2 120 E 20 65 invitrogen TOPO TA cloning kit for sequencing pCR2 1 50mg ml 1L 3 3 lml 50ug ml 90mm 15mm 1 20 25m1 Blue White 50ng ml 20mg m1 X gal in 32 4 1 l
35. igation sample 30 invitrogen TOPO TA cloning kit for sequencing 25yl ligation sample lul 4 2 heat shock 42 30 on ice 4 3 SOC kit 125n1 37 BEC 1 4 4 LB 1 125u1 25ul 4 5 14 18 P6R Blue White PCR 5 1 PCR primer M13 Reverse primer 5 CAG GAA ACA GCT ATG AC 3 M13F Foroward 20 primer 5 GTA AAA CGA CGG CA G 3 Reagent For 20u PCR Template DNA 2 10 X PCR buffer 2 ul 3 dNTP 2 5mM each 1 6 ul 4 primer 10pmol 0 4 ul 5 primer2 10pmol 0 4 ul 6 Taq polymerase 5U u 1 0 1 ul 1 dH 0 13 5 pl 5 2 Cycle 94 C 5 00 min 1 cycle J 94 C 0 30 min 52 C 0 30 min 30 cycles 129C X min J 72 C 7 00 1 cycle J 4 C hold 33 PCR 1000bp Imin 5 3 PCR
36. pH 8 0 Stock 1M 5 ml 100mM EDTA pH 8 0 Stock 0 5M 1 ml 10mM dH2O 44 mL 1M Tris HCl pH 8 0 autoclave Reagent Amount Final Conc Tirs 121 1 g 1M dH20 Up to IL HCl pH pH8 0 42m l 37
37. reparation of gradient gel DGGE 1 BIO RAD DCode system ITS2 PCR DGGE 8 acrylamide 30 60 gradient Sampayo et al 2007 Gradient gel 1 2 lt 3 gt 30 gt 12 o n gt 6 1 gt 7 8 DNA aoe Ww N 1 a 40 Acrylamide Bis 37 5 1 Reagent Amount Acrylamide 38 93 g Bis acrylamide 1 07 g dH2O Up to 100 0ml 0 45um pore LC 1 8 1 7 5ml RISE CHO b DCode Dye Solution
38. tfor2 5 GAA TTG CAG AAC TCC GTG 3 ITSrev2 5 GGG ATC CAT ATG CTT AAG TTC AGC GGG GT 3 25 8 1 PCR Reagent For 25u PCR Template DNA Gel or 2 ul 2 10 X PCR buffer 2 5 ul 3 dNTP 2 5mM each 2 ul 4 primerl 10 pmol 0 5 ul 1 primer2 10 pmol 0 5 ul 6 Taq polymerase 5U u 1 0 125 ul 1 dH 0 Up to 25 ul DGGE total 25ul PCR DGGE DNA RH TE 2ul 8 2 Cycle 94 C 5 00 min 1 cycle 94 C 0 30 min 52 C 0 30 min 35 cycles 72 C 1 00 min J ize 10 00 1 cycle J 4 C hold 8 3 PCR 8 4 PCR nano drop 26 4 Sequencing 4 1 Sequencing reaction Beckman 8880 2 K DTCS quick Start Master Mixy lt DNA template gt dsDNA PCR in DW 6 5ng 100bp plasmid 1 sequencing reaction Reagent Template DNA X ul 4 DTCS quick Start Master Mix 2 ul 3 5 X Seq buffer l ul 2 primer 1 6pmol 2 ul 1 dHLO Up to 10 ul 2 thermal cycle program 96 C 0 20 min 50 C 0 20 min 30 cycles 60 C 4 00 min l 4 C hold 3 3 1 stop solution

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