Wizard Plus SV Minipreps DNA Purification System No. TB281J 2001 7 A1270, A1330, A1340, A1460 A1470 Wizard Plus SV Minipreps DNA Purification System (a) DNA 30 20,000bp DNA Recombinant RNasin Ribonuclease Inhibitor (b,c) ( N2511in vitro DNA Wizard Plus SV Minipreps DNA Purification System 1 10ml DNA Wizard Plus SV Minipreps DNA Purification System DNA Tel. 03-3669-7980 prometec@jp.promega.com 1
Overnight Culture Centrifuge. Remove culture media Resuspend cells. Lyse cells. Neutralize. Clear lysate. Transfer lysate. Bind DNA. Wash, removing solution by centrifugation or vacuum. Elute plasmid DNA. Transfer Spin Column to a Collection Tube. Centrifuge. 1. Wizard Plus SV Minipreps DNA Purification System DNA 2 Tel. 03-3669-7980 prometec@jp.promega.com
Wizard Plus SV Minipreps DNA Purification System 50 A1340 (Vacuum Adapter 1-10ml 50 20ml Wizard Plus SV Cell Resuspension Solution 20ml Wizard Plus SV Cell Lysis Solution 30ml Wizard Plus SV Neutralization Solution 20ml Wizard Plus SV Column Wash Solution 50 Wizard Plus SV Minipreps Spin Columns 50 Collection Tubes (2ml) 550µ Alkaline Protease Solution 13ml Nuclease-Free Water 20 Miniprep Vacuum Adapters (d) Wizard Plus SV Minipreps DNA Purification System 250 A1470 (Vacuum Adapter 1-10ml250 75ml Wizard Plus SV Cell Resuspension Solution 75ml Wizard Plus SV Cell Lysis Solution 100ml Wizard Plus SV Neutralization Solution 100ml Wizard Plus SV Column Wash Solution 250 Wizard Plus SV Minipreps Spin Columns 250 Collection Tubes (2ml) 2,700µ Alkaline Protease Solution 25ml Nuclease-Free Water 20 Miniprep Vacuum Adapters Wizard Plus SV Minipreps DNA Purification System Wizard Plus System (e) Wizard Plus Wizard SV Plus Wizard Plus SV Minipreps DNA Purification System 50 A1330 250 A1460 Wizard Plus SV Minipreps DNA Purification System, Trial Size 10 A1270 1-10ml 10 3ml Wizard Plus SV Cell Resuspension Solution 3ml Wizard Plus SV Cell Lysis Solution 4ml Wizard Plus SV Neutralization Solution 4ml Wizard Plus SV Column Wash Solution 10 Wizard Plus SV Minipreps Spin Columns 10 Collection Tubes (2ml) 550µ Alkaline Protease Solution 1ml Nuclease-Free Water 5 Miniprep Vacuum Adapters A1330 A1460 Vacuum Adapter Wizard Plus SV Minipreps 22 25 Tel. 03-3669-7980 prometec@jp.promega.com 3
LB 14,000 g) LB 1.5ml (95%) (10,000 g) Wizard Plus SV Minipreps DNA Purification System Wizard Plus SV Column Wash Solution 10A1270)7ml 95% 11ml 50 A1330 A1340) 35ml 95% 55ml 250 A1460 A1470) 170ml 95% 270ml A600 2 4 DNA A. Luria-Bertani (LB) 1 10ml LB Terrific Broth Wizard Plus SV Minipreps LB 2. 3712 16 DNA DNA 5ml 5ml Wizard Plus SV Minipreps Spin Column DNA DNA DNA 10ml 10ml DNA 4 DNA 2 B. 1 5ml10ml 10,000 g 5 2. 250µl Wizard Plus SV Cell Resuspension Solution 1.5ml 1.5ml 250µl Wizard Plus SV Cell Lysis Solution 4 1 5 Alkaline Protease Solution4 5 Tel. 03-3669-7980 prometec@jp.promega.com
4. 10µl Alkaline Protease Solution 4 5 DNA 5. 350µl Wizard Plus SV Neutralization Solution 6. 14,000 g10 DNA Alkaline Protease Solution5 C. DNA Miniprep Vacuum Adapters Wizard Plus SV Minipreps DNA Purification System A1340 A1470) DNA Spin Column DNA Minipreps Vacuum AdapterVac-Man Laboratory Manifold) DNA Spin Column 2ml Collection Tube DNA 1. 850µ III.B Spin Column 2. 1 2ml Collection TubeSpin Column Collection TubeCollection Tube Spin Column 3. 95% 750µl Column Wash Solution Spin Column 4. 1 2ml Collection TubeSpin Column Collection Tube Collection Tube Spin Column (Vac-Man Vac- Man Jr. Laboratory Vacuum Manifold) Luer-Lok Stop Cock Miniprep Vacuum Adapter Spin Column Miniprep Vacuum Adapter 1. 850µl III.B 6 Spin Column 2. 15 Hg Spin Column Spin Column 3. 95% 750µl Column Wash Solution Spin Column 4.Spin Column Column Wash Solution Spin Column Spin Column Spin Column 1.5ml 5 10 S p i n Column Spin Column Hg 15 Hg 50.8 kpa 381 Torr 0.501 atm 7.37 psi 38.1 cm Hg 508 mbar Tel. 03-3669-7980 prometec@jp.promega.com 5
5. 250µl Column Wash Solution 62 7. Column Wash Solution Spin Column 1.5ml Spin Column Column Wash Solution 1 5. 250µl Column Wash Solution Spin Column 6 10 Spin Column 7Spin Column 2ml Collection Tube Spin Column Column Wash Solution 2 2ml Collection Tube DNA 11 TE 8. Spin Column 1.5ml 9. 100µl Nuclease-Free Water Spin Column DNA 1 10. DNA Spin Column 1.5ml 11.DNA 20 20 DNA 4DNA TE 100µl DNA 10µl 10 TE 12. 20 IV. A. Wizard Plus SV Minipreps DNA Purification System DNA DNA DNADNA Luria-Bertani(LB) 1 10ml LB 3712 16 A600 2.0 4.0 DNA 6 Tel. 03-3669-7980 prometec@jp.promega.com
B. 12kDa DNA enda enda1 EndA EndA EndA + 1. EndA EndA + EndA EndA + BJ5183 BL21(DE3) DH1 CJ236 DH20 HB101 DH21 JM83 DH5 JM101 JM103 JM110 JM105 LE392 JM106 MC1061 JM107 NM522 (NM EndA+) JM108 NM554 JM109 P2392 MM294 PR700 (PR EndA+) SK1590 Q358 SK1592 RR1 SK2267 TB1 SRB TG1 XL1-Blue Y1088 (Y10 EndA+) XLO BMH71 18 ES1301 enda1 enda EndA + Wizard Plus SV Minipreps DNA Purification System EndA + EndA DNA EndA + EndA EndA DNA EndA Tel. 03-3669-7980 prometec@jp.promega.com 7
C. EndA + EndA DNA Wizard Plus SV Minipreps DNA Purification System Alkaline Protease Solution subtilisin Carlsberg Bacillus licheniformis 1 1 250µg 2,3 ph9 4,5 DNA EndA DNA 0.2µg/µl 0.1µg/µl DNA DNA6DNA Wizard Plus SV Minipreps System pgem Vector (f) DH5 LB 1.5ml 3.5 5.0µg DNA DNA palter -1 Vector (f,g DH5 10ml LB 1.5 3.0µg DNA BigDye TM Wizard Plus SV Minipreps System ABI PRISM BigDye Terminator (Perkin-Elmer Corporation 4303149, 4303150, 4303151) BigDye Terminator (250mM Tris-HCl [ph 9.0], 10mM MgCl2) 6 BigDye Terminator 500 8 Tel. 03-3669-7980 prometec@jp.promega.com
2 BigDye Terminator BigDye Terminator BigDye Terminator 2 2. BigDye Terminator Terminator DNA 2 Nuclease-Free Water Terminator 2.5 5 2 4 6 8.0µl 4.0µl 2.0µl 1.3µl 200 500ng 200 500ng 200 500ng 200 500ng 3.2pmol 3.2pmol 3.2pmol 3.2pmol 0µl 2.0µl 3.0µl 3.4µl 20µl 20µl 20µl 20µl A600 2 4 10ml 5ml) Cell Resuspension Solution DNA Column Wash Solution Column Wash Solution DNA DNA DNA 10 Tel. 03-3669-7980 prometec@jp.promega.com 9
DNA 37 12 16 DNA DNA Lysis Solution Alkaline Protease Solution DNA Nuclease-Free Water DNA DNA DNA DNA Lysis Solution Column Wash Solution Wizard Plus Wizard SV Plus DNA DNA 70% DNA 10 Tel. 03-3669-7980 prometec@jp.promega.com
VI. 10 TE buffer 100mM Tris-HCl (ph 7.5) 10mM EDTA LB medium 10g casein peptone 5g yeast extract 5g NaCl 15g agar 1L 55 1 Wizard SV 96 Plasmid DNA Purification Systems Wizard SV 96 Plasmid DNA Purification System(a) 1 96 A2250 5 96 A2255 Wizard SV 96 Cell Resuspension Solution 500ml A7113 Wizard SV 96 Cell Lysis Solution 500ml A7123 Wizard SV 96 Neutralization Solution 500ml A1481 Wizard SV 96 Wash Solution 185ml A1311 Wizard SV 96 DNA Binding Plates 10 A2271 Wizard SV 96 Lysate Clearing Plates 10 A2241 Vac-Man Laboratory Vacuum Manifold 20 A7231 Vac-Man Laboratory Vacuum Manifold 2 A7660 Vac-Man 96 Vacuum Manifold 96 A2291 Vacuum Miniprep Adapters 20 A1331 SV Total RNA Isolation System(d) 50 Z3100 SV Total RNA Isolation System, Trial Size 10 Z3101 VIII. 1. Guntelberg, A.V. and Otteson, M. (1954) Compt. Rend. Trav. Lab. Carlsberg 29, 36. 2. Aehle, W. et al.(1993) Rational protein engineering and industrial application: structure prediction by homology and rational design of protein-variants with improved washing performance the alkaline protease from Bacillus alcalophilus. J. Biotechnol. 28, 31. 3. van der Osten, C. et al.(1993) Protein engineering of subtilisins to improve stability in detergent formulations. J. Biotechnol. 28, 55. 4. Vetter, R. et al. (1994) Highly alkaline proteases. U.S. Pat. No. 5,352,603. (October 4, 1994). 5. Shetty, J.K., Patel, C.P. and Nicholson, M.A. (1995) Method of preparation of purified alkaline protease. U.S. Pat. No. 5,439,817. (August 8, 1995). 6. Kahn, M. et al. (1979) Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Meth. Enzymol. 68, 268. Tel. 03-3669-7980 prometec@jp.promega.com 11
Wizard Plus SV Minipreps DNA Purification System: B 10,000 g 5 2. 250µl Cell Resuspension Solution 3. 250µCell Lysis Solution 4 4. 10µl Alkaline Protease Solution 4 5 5. 350µl Neutralization Solution 4 6. 10 C 1. Spin Column Collection Tube 1. Miniprep Vacuum Adapter 2. Spin Adapter Tube Spin Column Column 3. 2. Spin Column Collection Tube Spin Column 3. C 4. 750µl 4. 750µl Wash Solution Wash Solution 5. Collection Tube 5. 250µl Wash Solution Spin Colum 4 6. 250µl Wash Solution 6. 10 Spin Column 5 7. 2 7. Spin Column 2ml Collection Tube 2 C 8. Spin Column 1.5ml 9. DNA100µl Nuclease-Free Water Spin Column 1 10.DNA1.5mlSpin ColumnColumn 11.DNA 20 20TE 10µl 10 TE DNA TE 4 12 Tel. 03-3669-7980 prometec@jp.promega.com