Gel system in topic 11 doesn’t require denaturing. The buffer system has no reagent to denature the protein, so the separation is based on the net charge. The protein doesn’t change its shape while moving through the cell, it stays in its “native” state. Because we are under native conditions, the separation is based on charge. If we were denaturing the protein, then we would be concerned about molecular weight. What’s the diff. between normal hemoglobin and mutated hemoglobin in sickle cell patients? Normal hemoglobin has two more acidic residues, and in mutated version, they will be replaced by two less negative residues. Mutated will be more positive. In our gel system, pH of buffer is 9.2. The pI of the mutated hemoglobin would be slightly higher because it is less negative. Wild type pI is 6.8. A 6.8 protein in environment of 9.2 is a negatively charged molecule, so it will migrate to anode. Which will migrate further? The normal, more negatively charged one.
Amino acids: Don’t have to memorize each individual. On side chain of acidic and basic a.a.s, by looking at side chain, be able to predict if their pI is > or < 7?
Topic 5: What are the two blue dyes in the marker? Bromophenol blue (faster) and Xylan cyanol (slower). They help you to monitor the movement of the samples, and also help you with easy loading. They don’t intercalate the DNA samples. The dye that stains the DNA is the ethidium bromide, and it requires exposure on UV box.
Practicals: using the spectrophotometer. Will be given unknown samples, standard curve will be prepared for you. Know how to use prepared standard to obtain the concentration of the unknown. Second practical: electrophoresis: Demonstrate loading 10 μL samples. Then, set up electrophoresis using a prepared gel. This gel has wells set at the end of the gel. pI of samples will be given, and pH of buffer will be given, so you have to figure out how to connect the cables. Cathode is black and negative, anode is red and positive.
Calculation problems: Serial dilution, transformation efficiency, parallel dilution, know how to make certain percentage of gel. Beer’s law—follow unit carefully. Use the equation to solve for one component (in the report we solved for molar absorptivity).
What are the two functions of the nucleotides (dNTPs) in the PCR reaction? Building block of new DNA. ATP serves as energy source.
E8: Control group include two media: LB media spreaded with cell without pGLO. This is NOT a positive control. LB plate spreaded with –pGLO is TNTC plate. This tells you that all procedures and reagent did not harm the cells, or would affect transformation efficiency.
Another plate: LB/Amp spreaded with cell - pGLO. This is negative control. We should see no growth on this plate.
E8: List three different treatments: Calcium chloride, heat shock, last incubation/recovery time. To maximize transformation efficiency, a