Polymerase Chain Reaction

In week one Polymerase chain reaction (PCR) was used to amplify RTKs that were derived from thoroughly smashing five fruit flies and was combined with 1 ml Buffer A (630 ul ddH2O, 100ul 1M Tris/Hcl pH7.6, 200 µl 0.5M EDTA, 20 µl 5M NaCl, and 50 µl 10% SDS), then incubated for 15 minutes at 65˚C so that as much DNA could be secluded as possible. 200 ul of KAc/LiCl working solution, 1 part 5M KAc (potassium acetate) and 2.5 parts 6M LiCL (lithium chloride), mixed and chilled for a full 10 minutes. This important to do because this is how cell debris was separated from the nucleic acid. The solution was spun for 10 minutes at full speed in the microcentrifuge to create a supernatant that would be transferred into a new 250 ul tube. 150 ul of isopropanol that was at room were added vortexed and incubated again for 5 minutes at room temperature and once again spun at high speed for five minutes. …show more content…
Next the EtOH was removed leaving the pellet and allowing the pellet to dry by uncovering the test tube and placing it upside down in a kimwipe for five minutes at room temperature. Following that the pellet was resuspened in 50 ul of TE, and by tapping the pellet was dissolved. After it was place in the heat block for 1-2 minutes at 65˚C and continuously tap the pellet until it was completely dissolved to ensure that this DNA would react with the 1 ul PCR reaction. Later the nanodrop quantified the DNA amount of the sample and the yield should have been estimated to be around 3 ug from the 5