By Bimal D.M. Theophilus, Ralph Rapley
1Bimal D. Theophilus and Ralph Rapley offer organic and medical investigators with a entire number of new, fresh, and up-to-date PCR-based screening equipment appropriate for detecting the presence of either recognized and novel mutations. The tools hide aspect mutations (e.g., ASO-PCR, SSCP, DGGE, chemical cleavage), deletions (multiplex PCR, FISH, blotting), non-sense mutations (PTT), and extra. the hot and interesting thoughts of DNA array research, besides such lately constructed experimental tools as conformation-sensitive gel electrophoresis, also are integrated. each one bankruptcy explains the elemental thought at the back of the method and offers useful notes crucial for its profitable execution
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Additional info for PCR Mutation Detection Protocols
13. 6, 100 mM MgCl 2, 50 mM dithiothreitol. 14. 32P-γ-ATP: Specific activity > 3000 Ci/mmol. 15. T4 kinase: T4 polynucleotide kinase at 1 U/µL. Store at –20°C. 16. 0 mM ATP (freshly made from 20 mM stock). 3. 1. End-Labeling with Klenow 1. Resuspend 1–1000 ng of DNA in 42 µL of dH2O (see Note 2). Add 5 µL of 10X Klenow buffer, 1 µL of 32P-α-dNTP, 1 µL of dNTP mix, and 1 µL of Klenow. Incubate at room temperature for 15 min (see Note 3). 20 Harwood 2. Add 1 µL of dNTP chase. Incubate at room temperature for a further 15 min (see Notes 1 and 4).
Following transfer, the DNA is covalently crosslinked to the nylon membrane by exposure to ultraviolet irradiation, after which the blot may be stored or probed. From: Methods in Molecular Biology, vol. 187: PCR Mutation Detection Protocols Edited by: B. D. M. Theophilus and R. , Totowa, NJ 23 24 Rapley and Williams Fig. 1. A typical setup for capillary action Southern blot. 2. Materials 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Suitable apparatus for blotting, two buffer tanks, paper. Towels, Whatmann paper (see Fig.
13. 4 M NaOH and allow the DNA to transfer overnight. 14. After blotting, carefully remove the membrane and soak in 2X SSC to remove any adherent agarose. 15. Briefly blot dry the membrane, which is now ready for either storage at 4°C or immediate hybridization. 3. Hybridization and Autoradiography There are many different methods of hybridization that are dealt with elsewhere (6). However, after hybridization, the filter is probed with a 32P-labeled DNA fragment from the FVIII gene. 12, is a 647-bp StuI/SacI-restricted FVIII (7).