Cell Biology Lab
... of the transformed cells with the newly added genetic information grows overnight into a colony of millions of cells (National). The bacteria are then lysed and the plasmid DNA is removed from the bacterial cell. The DNA fragment can be cut out of the plasmid by using a restriction enzyme and then through electrophoresis (Alberts, 324-329). The restriction enzyme used to remove the DNA from the plasmid by cutting the DNA at a certain nucleotide sequence. Different restriction enzymes can be used for separating different gene sequences by each enzyme cutting at a different specific sequence of nucleotides. Once the restriction enzyme has cut the DNA then electrophoresis is used to separate the fragments according to size. The separated fragments form a pattern of dozens of parallel bands that reflect the composition of the DNA (Huskey). In electrophoresis, a solution containing a mixture of large molecules is placed on a conductive support, such as wet paper or a gel. An electric current is then applied to the solution, producing an electric field that causes the positively charged molecules to move in one direction and the negatively charged ones in another. In addition, the molecules of each substance in the solution have a specific charge and therefore move in the field at different rates. Eventually, the molecules are separated at different positions on the support. The current is then turned off, and the separated molecules are removed from the support. I hypothesize that pgkNEO is plasmid B and that digestion with enzyme BamHI will generate DNA fragments of 2500 bp (TTU) (Alberts, 324-329). II. Results Table 1 – These results show the distance the molecular weight markers traveled, along with the given bp, and the calculated Rf values of the markers. Standard ID # of BP Migration Rf Fragment 1 23,130 11 mm 0.20 Fragment 2 9,416 13 mm 0.23 Fragment 3 6,557 15 mm 0.27 Fragment 4 4,361 18 mm 0.32 Fragment 5 2,322 23 mm 0.41 Fragment 6 2,027 24 mm 0.43 Figure 1 – The graph shows the Rf values for the molecular weight markers that were given for the experiment by the TA. Trongeau 4 Table 2- Shows each digest that took place and the amount of plasmid, H2O, Buffer and enzyme that was added to each digestion. Reaction Tube 1 2 3 4 5 6 H2O 13 uL 13uL 12uL 12uL 11uL 11uL 10X Buffer 2 uL 2uL 2uL 2uL 2uL 2uL Plasmid A 5 u...