Lab 6 Molecular Biology Fatima Saavedra

Lab 6A
Background: E. Coli is an ideal organism for the molecular geneticist to manipulate and has been used many times in recombinant DNA research. E. Coli also contains plasmids, which also carry genetic information. The plasmids are extachromosomal and they exist separately from the chromosome. Hypothesis: The transformed E. Coli with the ampicilin resistant gene will be able to grow in the ampicilin plates, but not the transformed E. Coli will not. Materials: 2 sterile test tubes 250 L of ice cold 0.05M CaCl2 for each tube Large colony of E. Coli for each tube 10L of pAMP solution 250 L of Luria broth for each tube 100L of the cell suspension

Procedure: 1. Place two types of bacteria, with and without the plasmid, in solutions of just LB Agar and of LB Agar and Ampicilin. Data: LB + (Positive Control) LB- (Positive Control) LB/Amp+ (Experimental) LB/Amp- (Negative Control) Lawn Lawn 14 none

Error Analysis: Each step in the lab had a potential for error because each step was such a complicated process. All the measurements had to be very precise and accurate. An error could have been caused by CaCl2 due to the fact that it was frozen. Conclusion: The bacteria treated with the pAMP solution developed a resistance to ampicillin and were able to grow on the ampicillin+ plate. Those that were not treated with the pAMP were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would look in normal conditions. Therefore, the hypothesis is accepted.

Lab 6B
Background: Resistriction enzymes are essential tools in recombinant DNA methodology. Restriction enzymes recognize specific DNA sequences in double-stranded DNA and digest DNA at these sites. The result is the production of fragments of DNA of various lengths. Hypothesis: If lambda DNA HaeIII digest is run to establish a standard curve then the base pairs in lambda DNA digest/EcoRI can be estimated from the curve. Materials: Agorose Gel Phage lambda DNA digested with EcoRI Phage lambda DNA digested with HaeIII Digest Tracking dye, brome phenol blue Micropipette Buffer Electrical Supply Electrophoresis Chamber

Procedure: 1. Prepare the agar gel for the electrophoresis by microwaving it for the suggested amount of time. 2. When the gel has hardened, pour the running buffer over the gel and add the DNA samples into the wells. 3. Let the DNA run until the DNA is almost at the end of the gel. (Careful not to let it come out.) 4. Grab the stain and staining tray and let it stain for a while. 5. Put the gel in distilled water so the stain can be taken out of the gel. 6. Look and measure the gel and add the data into the data table.

Data:

Conclusion:

In conclusion, DNA fingerprinting is used to determine the size of the fragments that are cut by restriction enzymes. Restriction enzymes only cut at their specific protein recognition sites. This is useful because no two restriction enzymes code for exactly the same recognition site. From the data collected in the electrophoresis experiment, other sizes of parts can be hypothesized by following the size of the base pair to the line of best fit. This tells you about how many millimeters the base pair would probably go if allowed the same circumstances. Therefore the hypothesis is rejected.