Methods of the Bio Rad SmartSpec Plus Spectrophotometer Eric Hanson and Bilal Mohamed.

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Presentation transcript:

Methods of the Bio Rad SmartSpec Plus Spectrophotometer Eric Hanson and Bilal Mohamed

Abstract We were interested in characterizing the genetic diversity of Marchantia polymorpha by looking at their DNA. PCR (polymerase chain reaction) is used to amplify a segment of DNA, but is only necessary when there isn't enough DNA to run a comparative DNA analysis. This is the reason we were interested in checking if the Bio Rad SmartSpec was operating correctly. We used diluted stock lambda DNA (20ul DNA/80ul TE) as test solutions.

Introduction The Bio Rad spectrophotometer measures the amount of light a solution absorbs within a solution of extracted liverwort DNA. We were using it to determine which extraction kit produced the highest concentration of DNA.

Specs are important in analyzing DNA since you want to be working with enough sample to produce confident results. However, we were not able to test the extraction kits due to spec/human errors and the kits showed up late. Here are the methods we used to determine the usability of the spec.

Methods Made 100μl samples using different concentrations of λ DNA – 25, 50, 100, 150, 200, 250, and stock 300ng/μl Added 20 μl of dilute DNA and 80μl of TE to a disposable cuvette Set the spec to DNA/RNA assay Set to dsDNA Set A =50.00 μg/ml Blanked spec with 100μl of TE after every run Tested sample Set dilution factor to 5 (100μl/20μl )

Results

Conclusion The A260 readings and concentration readings were not consistent to the known concentration of the dilutions. Human and equipment error might be responsible for the inconsistency of the spec reading, either that or the conc. of the dilutions were not consistent One way to rule out stock concentration error would be to measure different DNA other than lambda