哈佛大学：《分子生物学》课程PPT教学课件（英文版）Techniques to manipulate DNA

Techniques to manipulate dna Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis dna hybridization Se outhern Blotting Melting tempe eratures PCR

Techniques to manipulate DNA Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis DNA hybridization Southern Blotting Melting temperatures PCR

Example: Is the brca3 gene involved in Suppressing breast cancer? BRCAl BRCA3 BRCA2 EcoRl bRCA ECoRl NIH

EcoRI BRCA3 EcoRI NIH Example: Is the BRCA3 gene involved in Suppressing breast cancer? BRCA1 BRCA2 BRCA3

Southern blotting ETOH ppt neno ProtK SDS Chloro tissue 9 Cleave with restriction enzymes Nitrocellulose Autoradiography Paper Towels Hybridize with labeled DNA Filter paper Nitrocellulose RNA probe: perform esIS autoradiography Capillary action transfers DNA from gel to nitrocellulose

Paper Towels Southern Blotting 1 tissue Phenol ProtK SDS Chloro ETOH ppt Spin

The"Hyperchromic Shift 0.4 0.3 0.2 220 240 280nm

The “Hyperchromic Shift” 2

Southern blotting ETOH ppt neno ProtK SDS Chloro tissue 9 Cleave with restriction enzymes Nitrocellulose Autoradiography Paper Towels Hybridize with labeled DNA Filter paper Nitrocellulose RNA probe: perform esIS autoradiography Capillary action transfers DNA from gel to nitrocellulose

Paper Towels Southern Blotting 1 tissue Phenol ProtK SDS Chloro ETOH ppt Spin

Agarose Gel Electrophoresis HO CHOH HO 3. 6-anhydro D-galactose L-galactose Sample well B offeR Glass plate Plastic block

Agarose Gel Electrophoresis _ + 3

Sample well 25 ng 1 kb ladder 0. 8% Agarose Agarose Gel Electrophoresis kb 864 Sybergold Detection Limit:0.2-0. 5 ng DNA EtBr Detection Limit: 5-10 ng dNA (Not in handout

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 High has no buffer: 0.5x TBE 0.5 ag/ml ethidium bromide electrophoresis: 1 VIcm for 16 hours resolving power at high Mw 10 kb 1 kb 3 Low %o has no 09%0 Resolving power 12 at low mw 100bp2 Distance migrated(cm)

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 TABLE 6.1 Range of Separation in Gels Containing Different Amounts of Agarose Efficient range of Amount of agarose in gel separation oflinear (%[w/v]) DNA molecules (kb) 0.3 5-60 0.6 1-20 0.7 0.8-10 0.9 0.5-7 1.2 0.4-6 0.2-3 2.0 0.1-2 Low Geling Agarose 4-6 0.02-0.4

Separation Range Vs. % Agarose 5 Low Geling Agarose 4-6 0.02-0.4

O H H H,C-C--C-NH, +CH,C--C-NH CH Acrylamide Methylenebisacrylamide S, 0(persulfate) 2 SO4.(sulfate free radical) CONH CONH TEMED CH2—CH—CH,CH一CH2CH (N,N, N, N-tetramethylenediamine) CONH CH CONH CH2CH—CH2CH—CH2-CH CONH CONH 7

TEMED (N,N,N,N-tetramethylenediamine) 7