Methods and Materials

DNA Purification and Restriction Map Analysis

50 µl of transformed and wild type cells were transferred to separate tubes containing 250 µl of Binding Buffer (Binding Buffer = 250 µl Guanidine hydrochloride, high salt, pH < 7). 300 µl from each tube was transferred to separate spin columns and centrifuged for 60 seconds.75 µl of Wash Buffer (Wash Buffer = 750 µl 80% Ethanol, high salt, pH < 7) was added to the spin columns and centrifuged for 60 seconds twice. (Testing the Hypothesis etc. 2015). The dried spin columns were transferred to tubes and 50 µl of Elution Buffer (Elution Buffer = 110 µl 10 mM Tris-HCl pH 8.5) was added (Testing the Hypothesis etc. 2015). 10 µl of purified Transformed and Wild Type PCR product were transferred to four tubes. 10 µl of unpurified PCR product was added to a no DNA control tube. 2.5 µl of 10 X Buffer + BSA (contains 1.5 µl 10 X Buffer + 1.0 µl 2 mg/ml BSA) was transferred to all of the tubes (Testing the Hypothesis etc. 2015). 2.5 µl of HindIII ((HindIII Enzyme @ 1U/ µl) was transferred into tubes Transformed and Wild type cells. 2.5 µl of TE ((10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA (pH 8.0)) was added to the Transformed, Wild Type, and no DNA control tubes (Testing the Hypothesis etc. 2015). All the tubes were incubated in a 37 o tempblock for 40 minutes. Afterwards 2 µl of STOP ((0.67% SDS, 26.7% sucrose, 0.067% bromophenol blue, 0.067 % xylene cyanol, 67 mM EDTA (8.0)) was added to the tubes (Testing the Hypothesis etc. 2015).

2015. Exercise 4: Testing the Hypothesis that DNA is the Genetic Material in Budding Yeast” DNA Purification and Restriction Map Analysis. Bioz 310, Biology Department, Virginia Commonwealth University.