The average of the two values was calculated as 255 x

This preview shows page 12 - 15 out of 23 pages.

number of colonies by the dilution factor and the dividing that by the volume in mL. The average of the two values was calculated as 2.55 x 10 8 (figure 4). This is the value for the average number of competent cells. The transformation efficiency (TE) was calculated using the number of colonies in tube 1. The TE was calculated to be 1500 transformants/ g of DNA (figure 3).
11 Discussion As mentioned in the results section, the results obtained from the agarose gel were inadequate. In figure 1, lane 5 and 6 represent pKan and pUC18 plasmids respectively. Bands were seen for the pKan plasmid, but however this was not the case for the pUC18 plasmid. If the plasmids had been extracted properly from their E.coli strains than bands would have appeared on the gel image. The extraction of pKan from its E.coli strain is proven by the presences of bands in the pKan lane. Two bands are visible in the pKan lane which indicates the presence of multiple size plasmids of pKan. The absence of bands in the pUC18 lane indicates inadequate extraction from the E.coli strain (DH5 ). This could have been due to experimental procedural errors. Two types of selection methods were used for this experiment, first the indirect (blue/white selection) method and second the direction selection method. As mentioned above the cells from tube 1 were spread onto a LB/amp/X-gal plate. The cells on the plate were transformed with uncut pUC18 plasmids. There was a growth of 3 colonies of which 2 were blue and 1 was white. There should have only been growth of blue colonies because uncut pUC18 plasmids contains a lacZ gene that is intact and capable of producing -galactosidase. X-gal on the plates is utilized by -galactosidase and hence is able to produce blue colonies. The growth of white colonies could be due to contamination and the uptake of another unknown DNA fragment into the multiple cloning sites of the lacZ gene. A way to eliminate contamination is to make sure that the surrounding areas and the equipment’s used are sterile. A different hockey stick should be used each time to spread the sample on the plates. The percent transformed for the uncut pUC18 was 3.92 x 10 -6 .
12 Cells from tube 2 were spread onto another LB/amp/X-gal plate. Cells from this plate were transformed with digested pUC18 plasmids. Growth of colonies from tube 2 were not obtained in this experiment. Growth of blue colonies should have been observed because the plasmids containing the lacZ gene and ori site are able to produce functional -galactosidase. This process was to make sure that digested pUC18 without ligation does not result in the lacZ gene being disrupted. Data provided by the instructor showed a formation of 45 blue colonies from the cells in tube 2. The growth of only blue colonies means that it was digested by restriction enzymes Hin DIII and Bam HI, but it was ligated back with no fragments in the multiple cloning site. The lacZ gene was still intact and was able to produce blue colonies. The restrictions enzyme cut up the lacZ gene but the pUC18 didn’t have a problem coming back to its original conformation. A study conducted by Norgard and others states that adding 5 mM of Mg 2+ to the wash solution and buffer can increase percent transformation more than 3-fold (Norgard et al., 1978). In the future Mg 2+

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture