PURESYSTEM classic II
Contents
1. 1 3 10 3 EGO PURESYSTEM custom2. PURESYSTEM custom info biocomber co jp Template DNA e Metal Affinity Resin PURESYSTEM Ribosome n Synthesized Protein 9 4 His Tagged Factors Step1 Addition of template DNA starts protein 3 synthesis reaction 4 Incubation at 37 Cfor 1 hour gt Step2 Addition of metal affinity resin for adsorption of tagged factors Incubation at 4 C for 1 hour EE Step3 Removal of ribosome and resin with tagged 9 factors by ultrafiltration membrane Vy Vy gt Synthesized protein can be isolated ea 1 PURESYSTEM 5 1 DNA 5 1 1 DNA PURESYSTEM DNA PCR DNA
3. 2 Forward Univ Primer 8 PCR DMSO TT 8 2 PCR 2 Forward Univ Primer 8 2 PCR 1 PCR Forward HT 5S GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA ATG Ni4 gt 0 PCR
4. SDS PAGE SDS PAGE RNA 20 30 kDa tRNA RNase 50
5. DMSO 7 2 PURESYSTEM DHFR 80 C 16 DNA DNA DNA T7 SD DNA
6. DNA DNA PURESYSTEM DNA DNA RNase EDTA EDTA PURESYSTEM RNA RNase RNase 1Y Step PCR Forward primer SD sequence ATG 5 Gene Sequence a 3 HIIIIIIIIIII ATT Reverse primer ATG TAA SD seduence 2 d Ste
7. ATG TAG TGA TAA T7 10 SD PCR 6 DNA PURESYSTEM PCR PCR ECR PURESYSTEM DNA 5 1 2 PCR DNA 2 PCR 2
8. DNA NR NR N C PURESYSTEM 2 1 1 Shimizu Y et al 2001 Nature Biotechnology vol 19 p 751 755 4 2 PURESYSTEM
9. PURESYSTEM classic Il 96 PURE2048C standard PURE2030C mini PURE2004C BioComber Sol A Sol B 1 6 Tf 8
10. DNA PURESYSTEM DNA 5 37 C 17 8 info biocomber cOo jDp
11. PURESYSTEM 4 1 PURESYSTEM 4 2 PURESYSTEM 5 1 DNA 5 2 5 3 5 4 DHFR 1 1 Sol A Sol B 2 96 mini standard Nuclease free Water Ll Ll Sol A 25 nl 300 ul Sol B 10 ul 200 ul Template DNA pl Total 50 ul 1000 ul 1 1 pmol PCR product or 0 5 ug plasmid DNA 2 20 pmol PCR product or 10 pg plasmid DNA 3 lt 4 37 C 1 6 2 2 1 PURESYSTEMY classic II 96 Prod PURE2048C
12. DNA DNA PURESYSTEM DNA Temp DHFR DHFR DNA DNA
13. RNaseA 3 75 pg 37 C 15 13 6 DHFR DHFR Dihydrofolate Reductase 1 2 17 18 SDS PAGE oe Sol A Sol B Temp DHFR Nucleas e free Water 12 5 ul Sol A 25 pul Sol B 10 ul Temp DHFR 2 S ul 0 S ug Total 50 ul 37 lt C 1 0 pl 100k 1 500xg
14. 2ml Ni NTA Agarose 1 10 10 ul 6 4 2C 1 7 1 5 ml 8 9 1 500xg 1 4 C 5 3 3 2 96 mini 1 1 30 pl 2 2ml 3 Ni NTA Agarose 1 10 10 ul 4 4 5C 1 100k 6 1 500xg 30 4 C 1
15. www biocomber co jp 18
16. PCR 4 1 PCR Universal Uniyv Primer 1 Reverse 2 PCR 5 PCR 2 PCR 1 70 PCR PURESYSTEM DNA 1 primer sequence Forward primer 5 AAGGAGATATACCA ATG Nj4 20 3 Reverse primer S TATTCA TTA Ni4 20 3 Universal primer GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCC Univ Primer CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA 3 5 1 3 DNA DNA DNA DNA T7
17. 2 Forward Univ Primer 2 pM 3 80 C 4 4 PURESYSTEM 4 1 PURESYSTEM PURESYSTEM 36 IF1 IF2 IF3 BEF Tu EF Ts EF G RF1 RF2 RF3 20 tRNA tRNA 70S T7 RNA tRNA NTP PURESYSTEM
18. 50 pl 96 Sol A Sol B Temp DHFR Univ Primer 96 x 25 pl 96 x 10 ul 2x5 pu 2x3500 ul 2 2 PURESYSTEM classic Il standard Prod PURE2030C 1 ml 3 Sol A Sol B Temp DHFR Univ Primer 3 x 300 pl 3 x 200 ul 1x 30 pl 1x 80 2 3 PURESYSTEM classic II mini Prod PURE2004C 50 pl 8 Sol A Sol B Temp DHFR Univ Primer Sol A Sol B Temp DHFR DHFR 8x25 nl Sx10 lx3 Lul 1 x 80 ul DNA 0 2 pg pl Univ Primer 2 PCR
19. 30 4 C 2ml Ni NTA Agarose 1 10 10 pl 11 4 12 13 14 15 16 C 1 1 5 ml 1 500xg 1 4 C 6 8 1 10 pl SDS PAGE 95 C 5 12 5 T 3 14 DHFR 20 1 14 1 1 2 DNA 3 4 UF 5 Ni NTA Agarose 3 DHFR 15 7 7 1 DNA 1 PCR
20. 1 PCR 2 PCR T7 2 PCR 1 PCR DNA DNA PCR 2 PCR DNA 1 Forward Reverse 2 1 9 2 DNA 1 1 PCR 3 PCR 7 1 PCR 2 PCR PCR PCR
21. 2 20 pmol PCR product or 10 pg plasmid DNA 3 lt 4 37 C 1 5 10 5 3 100 kDa 1 Ni NTA Agarose QIAGEN 100k Pall BIO RAD Vortex Mixer 5 3 2 1 96 7 mini 1 5s0 ul 2 100k 3 1 500xg 30 4 C 4
22. 1 7 5 3 4 3 standard 1 1 ml 200 pl 2 100k 3 1 500xg 30 60 4 C 4 1 2ml 5 Ni NTA Agarose 1 10 100 pl 6 4 C 1 7 2 ml 8 9 1 500xg 3 4 C 10 12 5 4 SDS SDS PAGE RI
23. p PCR Universal primer mR gt ATG TAA MAIIIUI T7 promoter ENN lt SD sequence Reverse primer T7 promoter SD sequence 2 2 PCR DNA 5 2 Temp DHFR 6 1 Sol A Sol B 2 96 mini standard Nuclease free Water Ll ul Sol A 25 ul 500 ul Sol B 10 ul 200 ul Template DNA ul Ll lt Total 50 ul 1000 ul 1 1 pmol PCR product or 0 5 pg plasmid DNA
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