7 BIOLOGY 242 : SPRING SEMESTER Figure 1.2: DNA Isolation Gel Electrophoresis Well 3 is mine. Figure 2: Gel Doc System Picture Figure 2.1: Gel Electrophoresis under UV light
Interpretation of Results: Semilog Graph Figure 2.2: Chart of Distances and Base Pairs M=DNA Marker L=Lambda Uncut P=PstI digest of DNA E=EcoRI digest of DNA H=HindIII digest of DNA Dista nce(m m) actual bp Dista nce (mm) Actual bp Dista nce (mm) Actual bp Distanc e(mm) Actua l bp Distanc e(mm) Actual bp Band 1 6m m 20,130 bp 6 mm 20,130 bp 10 mm 9,416 bp 8 mm 18,00 0 bp 6 mm 20,130 bp Band 2 10m m 9,416 bp - - 17 mm 3,200 bp 13 mm 5,300 bp 10 mm 9,416 bp Band 3 11m m 6,557 bp - - - - 14 mm 5,00 0 bp 13 mm 5,300 bp Band 4 16m m 4,361 bp - - - - 16 mm 4,361 bp - Band 5 - 2,322 bp - - - - - - - Band 6 - 2,027 bp - - - - - - - 8 BIOLOGY 242 : SPRING SEMESTER
Figure 2.3: Semilog Graph of Chart Above used to estimate bp for restriction fragments 9 BIOLOGY 242 : SPRING SEMESTER Semilog Graph Size in base pairs 100 1,000 10,000 100,000 Distance Traveled (mm) 6 10 11 16 18 20,130 9,416 6,557 4,361
Discussion Based from the results from out experiments we were able to conclude that what we had obtained was DNA and successfully were able to digest the genomic DNA and estimate band fragment sizes. We were very unsure whether we had obtained DNA during isolation because the pellet was barely visible. The spectrophotometer analysis allowed us to confirm our DNA concentrations. At first we did not get a positive reading most likely due to the small amount of sample I first put into the tube. I re-did the process and raised the sample amount to 20μl of sample and put it back into the spectrophotometer again and got a positive reading. For further confirmation, the analysis through gel electrophoresis showed the purity of the DNA sample. Proper well insertion of the sample was shown by a thick, single band, on the other hand if I had loaded the samples wrong a smear would have shown. Our next experiment which was our restriction digest analysis our group took my sample of DNA and cut it using a variety of restriction enzymes. Our agarose gel showed that lane 5 which was the sample cut with HindIII was similar in band length to the lambda DNA digested with HindIII which was our standard. Our EcoRI digest and PstI digest showed different band lengths and our uncut DNA was just one band. We created a table and a semi log graph that used the lambda DNA digested with HindIII as a standard curve since base pair length was known for all bands of the marker. However not all six bands were visible so we drew a best fit line based off of the first 4 bands. In addition, not every digest had 6 bands so we only estimated band length for those bands visible from the gel by measuring the distance of the bands traveled and using the best fit line for estimation. By repeating the experiment we may have been able to get better results on the digest analysis, maybe by adding more of the DNA sample in order to get thicker bands and possibly more bands on the gel analysis and fill in the blanks that we could not obtain. In addition, if we used pulse-
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