斯坦福大学分子生物学讲解之dna.pptVIP

Techniques to manipulate DNA PolyAcrylamide Gels What parameters affect theTm?(Tm is defined as the temp at which half the moleculesare single-stranded) Kinetics of Hybridization:Do I have time for a soda or a week-end in New York City? Results of Southern Blot Results of PCR #1 Results of PCR#5 PCR mutagenesis EcoRI HindIII Isolate products, mix Amplify digest, subclone sequence 20 PvuII * Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis DNA hybridization Southern Blotting Melting temperatures PCR BRCA3 EcoRI EcoRI NIH Example: Is the BRCA3 gene involved in Suppressing breast cancer? BRCA1 BRCA2 BRCA3 Paper Towels Southern Blotting 1 tissue SDS ProtK Phenol Chloro ETOH ppt Spin The “Hyperchromic Shift” 2 Paper Towels Southern Blotting 1 tissue SDS ProtK Phenol Chloro ETOH ppt Spin Agarose Gel Electrophoresis _ + 3 Sample Well 25 ng 1 kb ladder 0.8% Agarose 1 2 4 6 8 10 12 kb Agarose Gel Electrophoresis Sybergold Detection Limit: ~0.2-0.5 ng DNA EtBr Detection Limit: ~5-10 ng DNA (Not in handout) Log (MW) is linearly related to mobility 4 100 bp Low % has no Resolving power at low MW 1 kb 10 kb High % has no resolving power at high MW Separation Range Vs. % Agarose 5 Low Geling Agarose 4-6 0.02-0.4 TEMED (N,N,N,N-tetramethylenediamine) 7 8 DNA topology affects mobility of DNA 9 PFGE can resolve large DNA fragments 10 Paper Towels Southern Blotting 1 tissue SDS ProtK Phenol Chloro ETOH ppt Spin Helix Formation: Nucleation and Propagation 11 A-T A-T G-C Helix Melting 12 Tm depends on DNA length 13 % dsDNA Sigmoidal Curve: Highly Cooperative Melting! Effect of % GC Content on Tm 14 Effect of [Salt] on Tm 15 Predicting melting temperatures (see lecture notes) Cot Curves: Hybridization vs. Complexity 16 A Bi-Phasic Cot Curve 17 5 kb Repeats (20% total) 100 kb fragment Repeat DNA = 5 kb Longest non-repeat DNA = 100kb - 20 kb = 80 kb Difference in complexity = 80/5 = 16 Paper