nullTechniques to manipulate DNATechniques to manipulate DNAPhenol/chloroform extraction
Ethanol precipitation
Concentration determination
Gel Electrophoresis
DNA hybridization
Southern Blotting
Melting temperatures
PCRnullBRCA3EcoRIEcoRINIHExample: Is the BRCA3 gene involved in
Suppressing breast cancer? BRCA1BRCA2BRCA3nullPaper TowelsSouthern Blotting1nullThe “Hyperchromic Shift”2nullPaper TowelsSouthern Blotting1tissueSDSProtKPhenol
ChloroETOH ppt
SpinnullAgarose Gel Electrophoresis_+3nullSample Well
25 ng 1 kb ladder
0.8% Agarose124681012kbAgarose Gel Electrophoresis
Sybergold Detection Limit: ~0.2-0.5 ng DNA
EtBr Detection Limit: ~5-10 ng DNA(Not in handout)nullLog (MW) is linearly related to mobility4100 bpLow % has no
Resolving power
at low MW1 kb10 kbHigh % has no
resolving power
at high MWnullSeparation Range Vs. % Agarose5Low Geling Agarose 4-6 0.02-0.4nullTEMED
(N,N,N,N-tetramethylenediamine)7PolyAcrylamide GelsPolyAcrylamide Gels8nullDNA topology affects mobility of DNA 9nullPFGE can resolve large DNA fragments10nullPaper TowelsSouthern Blotting1tissueSDSProtKPhenol
ChloroETOH ppt
SpinnullHelix Formation: Nucleation and Propagation11nullA-TA-TG-CHelix Melting12What parameters affect the
Tm?
(Tm is defined as the temp at
which half the molecules
are single-stranded)What parameters affect the
Tm?
(Tm is defined as the temp at
which half the molecules
are single-stranded)nullTm depends on DNA length13% dsDNASigmoidal
Curve:
Highly
Cooperative
Melting!nullEffect of % GC Content on Tm14nullEffect of [Salt] on Tm15nullPredicting melting temperatures(see lecture notes)Kinetics of Hybridization:
Do I have time for a soda or a
week-end in New York City? Kinetics of Hybridization:
Do I have time for a soda or a
week-end in New York City? nullCot Curves: Hybridization vs. Complexity16nullA Bi-Phasic Cot Curve17nullPaper TowelsSouthern Blotting1tissueSDSProtKPhenol
ChloroETOH ppt
SpinResults of Southern BlotResults of Southern BlotControl TissueCancer TissueBRCA3EcoRIEcoRINIHnullBRCA3EcoRIEcoRINIHnullThe Polymerase Chain Reaction (PCR)1820-30Xnull94 C
(30 s)40-60 C
(30 s)72 C
(30-60 s/kb)MeltAnnealExtendCycle #2NIH20-30XResults of PCR #1Results of PCR #1Control TissueCancer TissueBRCA3EcoRIEcoRINIHPrimer Set #1Results of PCR#5Results of PCR#5Control TissueCancer TissueBRCA3EcoRIEcoRINIHPrimer Set #5nullPCR mutagenesisEcoRIHindIII20PvuIInullnullLinear
AmplificationGeometric
Amplification19nullhsp70EcoRIEcoRINIHExample: Is heat shock factor bound to
promoters before heat shock? EcoRIHSEHSFnullOptimal separation of fragments > 2 kb
requires low voltage0.5 10 V/cm6
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