Each tube was then flicked and spun in a microcentrifuge to mix the ingredients and bring them all together at the base of the tube. Next, the tubes were incubated at 37°C for 1 hour. While the tubes were incubating, 0.5 g of agarose were added to 50 mL of TAE buffer, which is a 1% mixture, to create an agarose gel. The solution was then microwaved to dissolve the agarose, placed in ice briefly to cool down, and then poured into the gel tray where it would solidify and take its shape. A 10 well comb was placed at one end to create 10 wells when the gel was done. When the digest tubes were done incubating, 7 µL of a mixture of 6x loading dye and SYBR green were added to each tube. They were mixed by pipetting up and down. TAE buffer was poured into the electrode chamber where the gel was. 11 µL of each mixture was then loaded into a well in this order: Ladder (1 KB Perfect made by Novagen), C, BglI, HindIII, B+H. The wells were next to the negative electrode so that the DNA would be 
pulled towards the positive. The electrode chamber was turned to 140 volts and ran for 45 minutes. When it was done, the gel was taken to a UV machine where it could be properly viewed and an attached printer printed a picture of the gel. The lane that the ladder was in was compared to a set standard for that ladder to tell me the number of base pairs each dash of DNA had. These were previously predicted using a website that does virtual electrophoresis experiments called http://tools.neb.com/NEBcutter2/.
	The gel (fig 1) turned out well. Each band of antimony is strong and it is easy to tell how many base pairs each one has. A standard curve was done (fig 2) to test the data. The drawn line fits moderately well with the data but it does not have great accuracy. Fig 8 gives some more information as to how the digests look and where the enzymes cut the plasmids. In order to make the final decision as to which plasmid antimony is, the predicted digests must be compared with the actual one (figs 3-7). By looking at figure 3, it can be seen that BglI and HindIII only cut the plasmid once, making figures 5, 6, and 7 not possible options to be antimony. Also, figure 1 shows that the plasmid only has around 3.3 Kbp which makes only pET17-b a logical selection for the identity of my plasmid. A final piece of evidence is that figures 3 and 4 are identical, which proves that the true identity of antimony is pET17-B.


I saw that he had left the balcony door open just a crack and knew that was where he would be. 