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Lab3-Sondagar%26Uppal-REVISED - Lab BCH2333 Section...

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Lab BCH2333 Section: TuA_b (Tuesday Afternoon) LABORATORY 3 Matias Alvarez FINAL GRADE = 88/100 Tuesday, February 10 th , 2009 Team 23 Shaheen Uppal 4918165 Priyen Sondagar 4868085
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Purpose In this experiment, the isolation and characterization of bacterial ( Escherichia coli ) DNA will be observed. In the first part of the experiment, DNA is going be isolated by adding different solvents which dissociate proteins and RNA from DNA. The different solvents that are added to help in isolating DNA are EDTA, SDS, NaClO 4 , and chloroform-isoamyl alcohol mixture. EDTA (ethylene-diamine tetraacetate) will break down the cell wall of the E.coli sample . SDS (sodium dodecyl sulfate) will binds to the proteins and coat them with a negative charge (the DNA backbone is negatively charged). This will cause there be repulsion between the DNA and protein and so they will dissociate. NaClO4 (a concentrated salt) weakens the DNA-protein interactions by minimizing the charge effects which are found in protein and DNA. The chloroform-isoamyl alcohol mixture will denature the protein even further and thus will cause the protein to coagulate between the organic and aqueous phase during centrifugation. The bacterial DNA sample will be incubated at a high temperature, which helps dissociate the DNA from the proteins as it denatures the proteins. In the second part of the first experiment, the isolated DNA will be tested for integrity and purity by measuring its absorbance at 234, 260, and 280 nm, using a UV spectrophotometer since DNA and proteins absorb UV light. The wavelengths and their ratios (A 234 /A 260 and A 260 /A 280 ) are indicators of purity of the DNA sample. The A 234 /A 260 ratio tests for protein contamination of DNA because the proteins will end up increasing the absorbance of the nucleic acids at 234 nm (But if the ratio is higher than 0.5 there is an indication of contamination). The A 260 /A 280 ratio is an indicator of RNA contamination with a range for the ratio being between 1.8 and 1.9 (If experimental ratio is higher than the range then there is indication of RNA contamination). In the second experiment, DNA melting curves will be constructed using different DNA samples (the in-lab prepared DNA sample and the commercial DNA samples). The DNA melting curves are indicators of the homogeneity and integrity of the DNA as well. Hyperchromic shift happens when the double helix of the DNA is separated to become a random-coiled single strand of DNA (this increases the absorbance at 260 nm). The DNA melting curves show the effect of heat on the absorption at 260 nm. From the curve, the melting temperature (Tm) can be defined. Every DNA species has a characteristic Tm and is used to characterize DNA samples. The effects of salt and denaturing agents on DNA melting curves will be observed in this experiment as well.
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Lab3-Sondagar%26Uppal-REVISED - Lab BCH2333 Section...

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