ENDEXTTM Technology
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1. 7 2 ENDEX WEPRO SUB AMIX 7 3 22 eCRES e CellFree Sciences E mail tech sales JP cfsciences com T 230 0046 75 1 201 TEL 045 500 2119 FAX 045 500 2117 Web site http www cfsciences com Y Endo and T Sawasaki 2004 High throughput genome scale protein production method based on the wheat germ cell free expression system J Struct Funct Genomics 5 45 57 T Sawasaki T Ogasawara H Morishita and Y Endo 2002 Acell free protein synthesis system for high throughput proteomics Proc Natl Acad Sci USA 99 14652 14657 T Sawasaki Y Hasegawa M Tsuchimochi N Kamura T Og2. 4 500 RNase mRNA PCR eCRES e CellFree Sciences 5 6 WEPRO 9240 SUB AMIX Y SGC WEPROY9240 o SUB AMIX YSGC 1 2 3 10 ul WEPRO 9240
3. SUB AMIX SGC 20 SUB AMIX SGC 4 15 24 5 1 9 4 8
4. e CellFree Sciences X SUB AMIX SGC um mH eCRES e CellFree Sciences 6 SDS PAGE SDS PAGE CBB NAERRSEBHSEEOD 1 3 ul DHFR 20 kDa kDa 250 A Molecular weight marker 150 B Negative control 100 C DHFR protein was expressed 20 Arrow indicates DHFR protein band 60 50 40 30 25 20 21 CES e CellFree Sciences 7 7 1 Premium PLUS Kit
5. 1 2 _ 3 4 5 e CellFree Sciences AA d C8 R SUB AMIX SGC eC e CellFree Sciences 5 PCR 5 1 1 pEU E01 MCS 2 step PCR SP6 E0O1 3 UTR 2
6. WEPRO 9240 d SUB AMIX SGC 1 2 2 3 3 10 ul WEPRO 9240 4 SUB AMIX SGC 11 SUB AMIX SGC 5 4 6 15 20 5 7
7. 030 099 4 1 50g im DHFR 1 0 ug ul Dihydrofolate Reductase 100ul 2nd PCR 1 uM et 100 ul 2nd PCR TTE T 10 nM ums 37532 ER ERIS FR OD N E ER S b HH Cj NIR ON 1 4 pEU E01 DHFR 0 2 ml PCR Tube fk SPU 0 2 ml PCR Tube Lid deSP6E01 0 2 ml PCR Tube ES Transcription 0 2 ml iui Tube Premix WEPRO 9240 0 2 ml PCR Tube SUB AMIX T rem strip SGC well CES e CellFree Sciences 3 3 3 3 1 DNA 4 2
8. 1 4 2 DNA polymerase 3 DNA polymerase PCR CES e CellFree Sciences 5 4 PCR 2nd PCR 1 5 ml 2 2nd PCR 2 5 100 1710 3 M pH 5 2 1 3 20 10 4 15 000 rpm 15 4 C 5 709 300 ul 6 15 000 rpm 5 4 C 7 15 2 8 2nd PCR 1 10 Nuclease free water 10 9 DNA2 ul 1 PCR
9. 3 pET 24 series pET 28 series PCR SPe pEU E01 DHFR PCR 4 PCR 30 CES e CellFree Sciences 5 2 1st PCR 5 2 1 1st PCR 1st PCR Cm us am 1st PCR bro duct c antisense primer XEF CDNA Primer 5 2 2 1st PCR gene specific primer bp 35 5 ccacccaccaccaccaATGNNNNNNNNNNNNNNNN 3 2nd PCR
10. 2nd PCR CS e CellFree Sciences 5 3 2nd PCR 5 3 1 2nd PCR T 2nd PCR deSP6E 01 1st PCR Product OPO 2nd PCR product 5 3 2 2nd PCR antisense primer 1st PCR antisense primer 5 2 2 X sense primer SPU deSP6E01 2 SP6 promoter 5 bp 100 ACCAATG SP6 promoter 3 E01 1st PCR ATG 5 ggtgacactatagAACTCACCTATCTCCCCAACACCTAATAACATTCA ATCACTCTTTCCACTAACCACCTATCTACATCACCACCCACCACC CES e CellFree Sciences 5 3 3 2nd PCR 1 0 2 m PCR Perm sk lasmMeNrp 4 om tuwspo s om Howesspeeeh 5 imw Teamewpme 5 mW emere em E Total 5 cycles 35 cycles 3 PCR 2
11. 3 TE DNA 1 0 ug ul 4 1 12 9 4 E01 Translational enhancer Xho 1 Xhoi DNA 260 nm 280 nm DEOH 1 70 1 85 DNA 4 2 DNA DNA 1 0 ug ul 0 05 ug ul CES e CellFree Sciences Multiple cloning site information EcoRV Spe 472 Saci Kpni Noti I POOO T DL O NEN 74 GATATCACTAGTTCTCGAGCTCGGTACCTGTCCGCGGTCGCGACGTACGCGGGCGGCCGCC CTATAGTGATCAAGAGCTCGAGCCATGGACA
12. 2 DNA CES e CellFree Sciences 5 5 PCR mRNA LT pEU E01 DHFR DHFR 1 Transcription Premix YRL IK E GEUE 21824 1 2 Transcription Premix DNA 2ul 2 3 37 4 3 4 1ul mRNA 4 1 Transcription Premix 2 pEU EO01 DHFR 1 0 ug ul 2 ul 3
13. 3 2 1 lt lt gt gt ENDEXT WEPRO 6869774 6905843 1 2
14. 4 1 25 24 1 pH 7 9 2 15 000 rpm 5 3 DNA 4 3 DNA 5 15 000 rpm 5 6 DNA 7 6 DNA 2 5 100 1710 3 M pH 5 2 8 20 C 10 9 15 000 rpm 4 20 10 70 800 ul DNA 11 15 000 rpm 4 10 12 13 DNA 10 20 14 DNA TE 15 DNA 260 nm 280 nm 260 nm
15. 3 37 C 1 3 4 1 ul mRNA 3 1 Transcription Premix 2 3 500 RNase mRNA DNA 4 2 mRNA A B A Molecular weight marker DNA B mRNA 196 agarose gel eCRES e CellFree Sciences 4 4 1 WEPRO 9240 amp SUB AMIX SGC
16. 4 1 DNA 422 DNA mRNA 37 C 1 4 3 15 C 20 4 4 SDS PAGE 6 e CellFree Sciences 2 2 PCR 2 step PCR DNA 5 2 5 3 DNA PCR 5 4 57 DNA mRNA 37 C 4 5 5 15 C 24 5 6 SDS PAGE 6 CES e CellFree Sciences 3 3 1 80 C 3 2 8 TS E01 MCS 0 2 ml PCR Tube 1 EE
17. 25241 gt 999 gt 99 709 TE 10 mM Tris 1 mM EDTA pH 8 0 Sterilized TE DNase RNase free water DEPC 3 3 2 PCR DNA 5 2 gene specific primer 1st PCR 5 2 2 antisense primer 1st PCR 2nd PCR 5 2 2 PCR PCR DNA Polymerase Buffer dNTP Ex7ag gt 99 70 DNase RNase free DEPC Exy7g9 eCRES e CellF
18. DNA 260 nm 280 nm DNA 1 16 TE DNA 1 0 ug ul 2 1 DNA 260 nm 280 nm 1 70 1 85 DNA 4 2 1 DNA EE DNA 1 0 ug ul 0 05 ug ul CES e CellFree Sciences 4 3 DNA mRNA pEU E01 DHFR DHFR 1 Transcription Premix YRL IK E GEUE 2 824 1 2 Transcription Premix DNA 1 0 ug ul 2 ul Hx
19. ENDEXT Technology Premium PLUS Kit Ver 1 00 Catalog No EDX PLUS CES e CellFree Sciences 1 kk 2 2 kk 2 2 1 DNA 2 2 2 PCR 3 3 T E A ONAE E 4 3 1 kk 4 3 2 A 4 3 3 DV C TASE E 5 3 3 1 DNA 5 3 9 2 PCR DNA Mii 5 4 DNA i 6 4 1 DNA 6 4 2 DNA nn 8 4 3 DNA mRNA kk 9 4 4 10 5 PCR 12 5 1 e 9 90 12 5 RERI 13 5 2 1 1st
20. PCR HMM 13 5 2 2 1st PCR 13 5 2 3 1st PCR 14 53 2NONROREREETETTNNTOTTTTET TKR0 80801 Oo 15 5 3 1 2nd PCR kk 15 5 3 2 2nd PCR 15 5 3 3 2nd PCR kk 16 5 4 PCR es 17 5 5 PCR mRNA OO RR st his iuuenes 18 5 6 EfNGECHUIBEISL IEOSBSRI esee entere nennen nennen nens 19 6 SDS PAGE 21 CO cda 22 7 1 22 EE so 22 7 3 22 eCRES e CellFree Sciences 1 Premium PLUS Kit Premium Expression Kit 2 DNA plasmd DNA PCR product PCR product plasmid 10 309 2 2 1 DNA DNA
21. GGCGCCAGCGCTGCATGCGCCCGCCGGCGG Bamlil Sma Sall Ncol o ATAAATTGGATCCATATATAGGGCCCGGGTTATAATTACCTCAGGTCGACGTCCCATGG 193 TATTTAACCTAGGTATATATCCCGGGCCCAATATTAATGGAGTCCAGCTGCAGGGTACC DEU E01 MCS 3 747 bp Amp pEU E01 MCS sequence SP6 promoter 17 1 Translational enhancer E01 16 72 Multiple Cloning Site 74 193 Origin 1190 1830 Ampicillin resistance gene 1974 2838 1 SP6 G ATTTAGGTGACACTATAG CES e CellFree Sciences 4 2 DNA DNA DNA DNA 1 500 bp 4 3 1 DNA 1 DNA
22. asawara and Y Endo 2002 A bilayer cell free protein synthesis system for high throughput screening of gene products FEBS Lett 514 102 105 K Madin T Sawasaki T Ogasawara and Y Endo 2000 A highly efficient and robust cell free protein synthesis system prepared from wheat embryos Proc Natl Acad Sci USA 97 559 564 Premium PLUS Kit J ver 1 0 Oct 26 2011 02011 CellFree Sciences Co Ltd All rights reserved 23
23. deSP6E01 ATG NNN 20 antisense primer 1st PCR 2nd PCR TES 1 6 kb 20 2nd PCR CES e CellFree Sciences 5 2 3 1st PCR 1 0 2 ml PCR Template plasmid DNA 250 pg ul Mdemeteewaer 3035 ma 77 29 2 Thermal Cycler 35 cycles 3 PCRZEY2yut 72Zmn AZIJI V amp SXSIL BnedmmmAOo 2 2bh 4 1 10 2 DNA polymerase 3 DNA polymerase PCR 4 PCR
24. ree Sciences 4 DNA 4 4 DNA 1 pEU E01 MCS cDNA 1 2 o pEU E01 MCS 7 SP6 E01 cDNA pEU E01 MCS Bl QIAGEN DNA QIAGEN Plasmid Midi Kit Plasmid Maxi Kit DNA 260 nm 280 nm TOR 260 nm DNA 260 nm 280 nm DNA
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