PCR 기초강좌10 - RNA 에 의&ia

PCR 기초강좌10 - RNA 에 의&ia

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: 100 nM Cy3TM-dUTP 1 2 3 4 5 6 7 8 9 : : : : : : : : : 100 nM Cy5TM-dUTP 9 8 7 6 5 4 3 2 1 2 Life Science & Biotechnology No.11 660 nm signal(Cy5) 570 nm signal(Cy3) Cy5 concentration/Cy3 concentration Life Science & Biotechnology No.11 3 G A C G T A C G T A C G T G T C A A G A G G 37C 50C 65C 4 Life Science & Biotechnology No.11 G A C G T G T A A A G A G G A C G T G G T C A A G A G G G A C G T G T C C A G A G G A C G T G G T C C A G A G G Life Science & Biotechnology No.11 5 BIOMEDICALS Tel. 02-577-2002 Fax. 02-577-3691 E-mail bohan@bohan.co.kr URL www.bohan.co.kr GMS 417 Arrayer Sample Slide glass GMS 418 Array Scanner Y Y Life Science & Biotechnology No.11 7 <pAUR135 > Acc III, Afl II, Aor51H I, Bal I, Bgl II, Bln I, Bpu1102 I, BssH II, BstX I, Cla I, Cpo I, Eco52 I, Eco81 I, Fba I, Hind III, Mlu I, Nae I, Not I, Nru I, Nsp V, PmaC I, PshA I, Sac II, Sal I, Sfi I, SnaB I, Spe I, Tth111 I, Van91 I, Xba I, Xho I 8 Life Science & Biotechnology No.11 (A) 1 2 3 4 (B) 1 2 3 4 1 3 2 4 9.7 kb 9.7 kb 2.4 kb 1 6 2 3 4 5 YPD AbA/YPD (2 g/ml) 1) Hashida-Okado, T., Ogawa, a., Kato, I. and Takesako, K. (1998) FEBS Letters 425, 117-122. 2) Hashida-Okado, T., Ogawa, A., Endo, M., Takesako, K. and Kato, I. (1996) Mol. Gen. Genet. 251, 236-244. 3) Akada, R., Yamamoto, J. and Yamashita, I. (1997) Mol. Gen. Genet. 254, 267-274. 4) Kawahata, M., Amari, S., Nishizawa, Y. and Akada, R. (1998) Yeast 14, in press. 5) Akada R. (1998) 93, 113-119. Q&A Q1 A1 Q2 A2 Life Science & Biotechnology No.11 9 Jun Yasuda 10 Life Science & Biotechnology No.11 Life Science & Biotechnology No.11 11 Case 1 Case 2 Case 3 Case 4 N N T T N N T T N N T T N N T T (bp) 2k A B1 B0 B C D E F G H I J K L M N 710 489 O P Q 404 R 325 S 12 Life Science & Biotechnology No.11 1) Welsh, J., McClelland, M. : Nucl. Acids Res., 18, 7213-7218 (1990) 2) Welsh, J., McClelland, M. : in The polymerase chain reaction (ed. Mullis, K. B., Ferr, F., Gibbs, R. A.), pp. 295-304, Birchaser, Boston (1994) 3) Sorbral, B. W. S., Honeycutt, R. J. : in The polymerase chain reaction (ed. Mullis, K. B., Ferr, F., Gibbs, R. A.), pp 304319, Birchaser, Boston (1994) 4) Ionov, Y., Peinado, M. A., Malkhosyan, S., Shibata, D., Perucho, M. : Nature, 363, 558-561 (1993) 5) Kohno, T., Morishita, K., Takano, H., Shapiro, D. N., Yokota, J. : Oncogene, 9, 103-108 (1994) 6) Peinado, M. A., Malkhosyan, S., Velazquez, A., Perucho, M. : Proceedings of the National Academy of Sciences of the United States of America, 89, 10065-10069 (1992) 7) Kubota, Y., Shimada, A., Shima, A. : Mutation Research, 283, 263-270 (1992) 8) Perucho, M., Welsh, J., Peinado, M. A., Ionov, Y., McClelland, M. : in Methods in Enzymology, 254, pp. 275290, Academic Press, Orland (1995) 9) Williams, J. G., Kubelik, A. R., Livak, K. J., Rafalski, J. A., Tingey, S. V. : Nucl. Acids Res., 18, 6531-6535 (1990) 10) Siebert, P. D., Larrick, J. W. : Nature, 359, 557-558 (1992) 11) Schierwater, B., Ender, A. : Nucl. Acids Res., 21, 4647-4648 (1993) 12) Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D., Gelfand, D. H. : PCR Methods & Applications, 2, 275-287 (1993) Life Science & Biotechnology No.11 13 Hiroyuki Aburatani, Tatsuhiko Kodama " " 14 Life Science & Biotechnology No.11 ' Life Science & Biotechnology No.11 15 16 Life Science & Biotechnology No.11 (a) (b) Life Science & Biotechnology No.11 17 1) Lisitsyn, N., Lisitsyn, N., Wigler, M. : Science, 259, 946-951 (1993) 2) Lisitsyn, N. A. : Trend in Genet., 11, 303-307 (1995) 3) Jonsson, J., Weissman, S. : Proc. Natl. Acad. Sci. USA, 92, 8385 (1995) 4) Rosenberg, M., Przybylska, M., Straus, D. : Proc. Natl. Acad. Sci. USA, 91, 6113-6117 (1994) 5) Chang, Y., Cesarman, E., Pessin, M., Culpepper, J., Knowles, D., Moore, P. : Science, 266, 1865-1869 (1994) 6) Lisitsyn, N., Segre, J., Kusumi, K., Lisitsyn, N., Nadeau, J., Frankel, W., Wigler, M., Lander, E. : Nature Genet., 6, 57-63 (1994) 7) Lisitsyn, N., Lisitsyn, N., Dalbagni, G., Barker, P., Sanchez, C., Gnarra, J., Linehan, W., Reid, B., Wigler, M. : Proc. Natl. Acad. Sci. USA, 92, 151-155 (1995) 8) Schutte, M., Da Costa, L., Hahn, S., Moskaluk, C., Hoque, A., Rozenblum, E., Weinstein, C., Bittner, M., Meltzer, P., Trent, J., Yeo, C., Hruban, R., Kern, S. : Proc. Natl. Acad. Sci. USA, 92, 5950-5954 (1995) 9) Drabkin, J., Mendez, M., Rabbitts, P., Varkony, T., Bergh, J., Schlessinger, J., Erickson, P., Gemmill, R. : Genes Chromosomes Cancer, 5, 67-74 (1992) 10) Kamb, A., Gruis, N., Weaver-Feldhaus, J., Liu, Q., Harshman, K., Tavtigian, S., Stockert, E., Day, R. R., Johnson, B., Skolnick, M. : Science, 264, 436-440 (1994) 11) Hubank, M., Schatz, D. : Nucl. Acid. Res., 22, 5640-5648 (1994) TaKaRa Taq TaKaRa Ex Taq TaKaRa LA Taq 18 Life Science & Biotechnology No.11 24 Life Science & Biotechnology No.11 1) Koyama, N. et al. (1991) J. Immunological Methods 139, 17-23. 2) Miura, M. (1998) The BONE 12, No. 4, 47-51. Life Science & Biotechnology No.11 25 26 Life Science & Biotechnology No.11 1) (1997) 2) Sato, N. et al. (1997) , 72, 801-805. 3) Muratsubaki, H. (1987) J. Biochem., 102, 705-714. 4) Hashida-Okado, T., Ogawa, A., Kato, I. and Takesako, K. (1998) FEBS Letters, 425, 117-122. 5) Asano et al. (1998) p41. Life Science & Biotechnology No.11 27 + plasmid - plasmid 28 Life Science & Biotechnology No.11 1) Kuroda, M. et al. Mol. Gen. Genet. (in press). 2) Gems, D. et al. (1991) Gene 98, 61-67. 3) Tada, S. et al. (1991) Mol. Gen. Genet. 229, 301-306. Life Science & Biotechnology No.11 29 [cDNA] 1000 100 10 1 A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1000 100 10 1 E F G H Human Rapid-ScanTM Panel 1. 5. 9. 13. 17. 21. Brain Liver Muscle Salivary Gland Ovary PBL 2. 6. 10. 14. 18. 22. Heart Colon Stomach Thyroid Gland Uterus Bone Marrow 3. 7. 11. 15. 19. 23. Kidney Lung Testis Adrenal Gland Prostate Fetal Brain 4. 8. 12. 16. 20. 24. Spleen Small Intestine Placenta Pancreas Skin Fetal Liver Mouse Rapid-ScanTM Panel 1. 5. 9. 13. 17. 21. Brain Thymus Muscle Adrenal Gland Embryo e8.5 Breast/Virgin 2. 6. 10. 14. 18. 22. Heart Liver Lung Ovary Embryo e9.5 Breast/Pregnant 3. 7. 11. 15. 19. 23. Kidney Stomach Testis Uterus Embryo e12.5 Breast/Lactating 4. 8. 12. 16. 20. 24. Spleen Small Intestine Skin Prostate Gland Embryo e19 Breast/Involuting 30 Life Science & Biotechnology No.11 [cDNA] 1000 100 10 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 GENE(tissue expression) -ACTIN (ubiquitous) 1000 100 10 1 -ACTININ 2 (muscle/heart/brain) 1. 2. 3. 4. 5. 6. Brain Heart Kidney Spleen Liver Colon 7. 8. 9. 10. 11. 12. Lung Small Intestine Muscle Stomach Testis Placenta 13. 14. 15. 16. 17. 18. Salivary Gland Thyroid Gland Adrenal Gland Pancreas Ovary Uterus 19. 20. 21. 22. 23. 24. Prostate Skin PBL Bone Marrow Fetal Brain Fetal Liver Life Science & Biotechnology No.11 31 32 Life Science & Biotechnology No.11 1. Probe Liquid migration Hybridization; 15 min/37 Biotin-labeled ssDNA 2. Binding of conjugate; 5 min/Room Temperature Streptavidin-AP 3. Enzymatic color reaction; 7 min/Room Temperature Chromogenic Substrate 4. Stop reaction; 3 min/Room Temperature Stop Solution Sample Stop Solution Chromogenic Substrate Streptavidin/AP Conjugate Empty Life Science & Biotechnology No.11 33 34 Life Science & Biotechnology No.11 FN(ng/ml) No. 1 : y=29056.524x + 14.203 No. 2 : y=17067.014x + 14.847 No. 3 : y=26491.142x + 11.890 r=0.992 r=0.996 r=0.998 1) Katayama, M. et al. (1989) Exp. Cell. Res. 185, 229. 2) Katayama, M. et al. (1991) Clin. Chem. 37, 466. 3) Katayama, M. et al. (1993) Clin. Chim. Acta. 217, 115. UFN per creatinine (unit/microgram creatinine) Life Science & Biotechnology No.11 35 36 Life Science & Biotechnology No.11 38 Life Science & Biotechnology No.11 Life Science & Biotechnology No.11 39 pET GST Fusion Systems 41 and 42 GST Bind Resin 1. Smith, D. B. and Johnson, K. S. (1988) Gene 67, 31-40 40 Life Science & Biotechnology No.11 Perfect DNATM Markers Protein Refolding Kit Life Science & Biotechnology No.11 41 42 Life Science & Biotechnology No.11 ProSieve Color Protein Markers F50000 F50001 F50010 F50011 F50021 F50031 F50041 F50070 F50071 F50080 F50081 F50090 F50091 F50101 F50111 F50152 F50181 F50513 F50611 F50615 F53440 F53734 F54711 SeaKem LE Agarose SeaKem LE Agarose SeaKem ME Agarose SeaKem ME Agarose SeaKem HE Agarose SeaKem HEEO Agarose SeaKem HGT Agarose SeaKem GTG Agarose SeaKem GTG Agarose NuSieve GTG Agarose NuSieve GTG Agarose NuSieve 3:1 Agarose NuSieve 3:1 Agarose SeaPlaque Agarose SeaPlaque GTG Agarose SeaKem GOLD Agarose MetaPhor Agarose SYBR GREEN I 50% Long Ranger Solution 50% Long Ranger Solution GELBOND FILM 0.1 mm GELBOND FILM 0.2 mm GELBOND PAG FILM 0.2 mm 125 g 25 g 125 g 25 g 25 g 25 g 25 g 125 g 25 g 125 g 25 g 125 g 25 g 25 g 25 g 25 g 25 g 10 X 50 250 ml 1 102 mm X 16.5 m 85 X 100 mm (100 ) 138 X 158 mm (50 ) 252,000 60,000 285,000 72,000 78,000 87,000 87,000 369,000 81,000 483,000 150,000 462,000 144,000 171,000 264,000 162,000 165,000 300,000 255,000 702,000 246,000 123,000 183,000 223,000 54,000 247,000 64,000 71,000 76,000 69,000 323,000 74,000 394,000 115,000 394,000 118,000 162,000 191,000 140,000 154,000 191,000 154,000 559,000 162,000 86,000 120,000 12% 10% 13% 11% 9% 13% 21% 12% 9% 18% 23% 15% 18% 5% 28% 14% 7% 36% 40% 20% 34% 30% 34% 43 Life Science & Biotechnology No.11 44 Life Science & Biotechnology No.11 Life Science & Biotechnology No.11 45 Q1 A1 Q2 A2 Q3 A3 Q4 A4 Q5 A5 Q6 A6 Q7 A7 Q8 A8 46 Life Science & Biotechnology No.11 NEW PRODUCTS Life Science & Biotechnology No.11 47 www.bohan.co.kr 48 Life Science & Biotechnology No.11 Life Science & Biotechnology No.11 49 NuSieve 3:1 agarose Turn your PCR into a work of art. 622 bp - 238-242 bp - 110 bp - Why settle for less than state of the art? With NuSieve 3:1 agarose, you can create PCR images of extraordinary sharpness and clarity. First time, every time. NuSieve 3:1 agarose is performance tested for fine resolution of DNA fragments agarose and create your own little masterpiece. 1,000 bp. This molecular biology grade agarose is easy to pour at high concentrations and offers speed and convenience. Work with NuSieve 3:1 NuSieve 3:1 agar ose Go straight to the source Visit us at http://www.bioproducts.com BIOMEDICALS Tel. 02-577-2002 Fax. 02-577-3691 E-mail bohan@bohan.co.kr URL www.bohan.co.kr New PCR*Reaction "Soup" of PCR Products with Heterogeneous Termini T ypical PCR conditions generate a mixture of products with heterogeneous termini.1,2 This heterogeneity results in difficulty ligating PCR products to either blunt or dT-tailed vectors3 for cloning purposes. Novagen's new Perfectly BluntTM Cloning Kits simplify cloning of any DNA fragment regardless of composition of condition of 3' termini; blunt ends, 3' overhangs, or ragged ends ligate with equal ease and greater cloning efficiency(3-fold to 24-fold higher). The kits contain everything needed for end conversion, ligation, and transformation. End Conversion Reaction Heat Inactivation (5 minutes) Homogeneous Products, Blunt and Phosphorylated Ligation Reaction References 1. Brownstein, J. M., et al. (1996) BioTechniques 20, 1004-1010. 2. Magnuson, V. L., et al. (1996) BioTechniques 21, 700-709. 3. Novy, R. E., Yaeger, K. W., and Kolb, K. M. (1996) InNovations 6, 7-11. *The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. Conditions that use DNA polymerases lacking 3' 5' exo-activity(e. g., Taq, Tth ) Perfectly Blunt Vector Insert is combined with ready-to use vector and ligated(15 minutes). Subsequent transformation into NovaBlue Competent Cells generates recombinant colonies that are visualized easily by blue/white screening www.novagen.com e-mail: novatech@novagen.com TaKaRa Z-Taq TM BIOMEDICALS BIOMEDICALS Tel. 02-577-2002 Fax. 02-577-3691 E-mail bohan@bohan.co.kr URL www.bohan.co.kr ...
View Full Document

Ask a homework question - tutors are online