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
