Answer Happy

mat d Painter Font 154 G What is the purpose of adding the loading dye to the samples? 3. DNA size standards are prepared by adding loading dye to 10 μl of a DNA ladder solution. Paragraph GEL LOADING QUESTION 7: What is it necessary to have DNA size standards when using agarose gel electrophoresis? 127 1. The pre-poured agarose gel (keeping it on its tray) is placed into the gel box. Be sure that it is properly situated and level in the gel box. "Make sure that the wells are nearest the negative electrode of the gel bax. QUESTION 8: Why is it important to have the wells nearest the negative electrode of the gel box? 2. Running buffer is added to the gel box until the gel is covered by at least 2 mm of loading buffer. QUESTION 9: What is the negative control sample? 3. In Part I of the analysis of Anolis pathogens lab, 2 different unknowns (Unknown 1 and Unknown 2) Anolis DNA samples were analyzed in duplicate (yielding 4 total PCR reaction samples). Also included in the PCR samples is one positive control reaction and one negative control reaction. QUESTION 10: What is the positive control sample?

4. Given the above, there are 7 samples total (2 unknown samples in duplicate, 2 controls, and 1 DNA standard). In the diagram of the gel on the last page (which 8 wells), label which sample you would add to each well. 5. Samples are loaded by adding 20 μl of each sample to the appropriate well. For the DNA standards, only 10 μl is loaded. RUNNING THE GEL 1. Make sure the power supply is off. 2. Snap on the gel box lid being sure to put positive to positive (red) and negative to negative (black). 3. Turn on the power to the power supply. 4. Set to constant voltage. 5. Set voltage to 100 volts. 6. Press the run button (dude running symbol) 7. Run for 30-60 minutes. RESULTS 1. On gel diagram on the last page, label which end would be near the positive electrode of the gel box, and which would be near the negative electrode. 2. Assume that the DNA ladder that was used as DNA standards includes bands of 50 bp, 100 bp, 200 bp, 400 bp, 600 bp, 800 bp, 1000 bp, 1500 bp, and 2000 bp. On the gel diagram, in the DNA standards lane, draw in bands for each of the size standards listed above. 3. Considering that a positive PCR product from plasmodium DNA should be approximately 500 base pairs (bp) long: Draw in the expected bands for the positive and negative control lanes. 4. Assuming that Unknown Sample 1 contained DNA from the malaria pathogen, but Unknown Sample 2 did not contain DNA from the malaria pathogen: Draw in the expected bands for all 4 unknown sample lanes.