FORDHAM BISC 1001 - Lab Exercise 3

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Isolating and Analyzing DNA with Several Lab Methods 2 28 24 Purpose Methods The purpose of this lab is to isolate DNA quantify it and then use several methods including PCR sequencing and Spa typing Finding the exact strain and species will help determine the best route of treatment Sample s tested Staphylococcus aureus Isolate Protocols DNA isolation was done through DNA extraction and is a three step process The cells are lysed with proteinase K through a multi step process I ll note that when I pipetted my mixture into the QIAamp Mini spin column a lot of the mixture got stuck in a foam in the pipet After lysing the cells chemically they re also heat treated The next step was to purify the DNA in spin columns along with altering the wash solutions to remove any waste Finally the DNA was eluted so we could collect unbound DNA To determine how much DNA was isolated it was quantified with spectrophotometry The amount of DNA quantified was used to determine the correct ratio used for the PCR The PCR solutions were then prepared accordingly Results DNA Quantitation The A260 Value 0 024 The A260 A280 ratio 1 24 There was 9 4 ug ml of DNA extracted 9 4 V1 1 50 V1 5 319 5 319 27 H2O 17 681 H2O Conclusion Discussion Knowing the total volume of DNA H2O and the master mix is 50 microliters the C1V1 C2V2 method was used to find the initial volume of DNA A conclusion will be updated in the next laboratory entry because spa typing has not been conducted yet Next Steps The purpose of spa typing is to identify specific strains of Staphylococcus aureus strains We re using this technique after our PCR and sequencing to get a complete identification of our isolate That way it can be determined whether the Staph is MRSA or MSSA


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FORDHAM BISC 1001 - Lab Exercise 3

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