E. Coli and D. Aquaticus PCR Reactions with Electrophoresis Gel

 This week was spent working productively on PCR reactions for both E. Coli and D. Aquaticus. The plan is to do a transformation with the cr2 gene, taken from the E.Coli using the pRAD1 vector in D. Aquaticus. We then ran gels, using electrophoresis to confirm the ~500bp and 7612bp sections

METHOD

 Multiple flask of TGY and LB+amp broth were made for our bacteria along with plates. Both were incubated at 37C for 24 hours. For E. Coli PCR, 50ul of culture was incubated at 95C for 10 minutes in an eppendorf tube. 4ul of culture were used and 2ul of Forward and Reverse Primers were added to the tube, along with 32ul of Master Mix. We then split this into 2x 20ul reactions for PCR. Thermocycler settings are shown in the photo below. 

For our gel and confirmation of our 500bp cr2 gene, we ran a electrophoresis gel. This contained 2ul of PCR sample, 8ul of dH20 and 2ul dye. We then ran this with a 100bp ladder at 80Ma for roughly 15 minutes. See photo below

For our D. Aquaticus PCR, our fragment was much larger, around 7200bp so a longamp PCR was performed. For this PCR, 46ul of Master mix was used along with 4ul of culture DNA. Two separate reactions were also ran this time, both boiled for 15 minutes at 95C. Longamp PCR settings were as follows; Initial Denature 94C for 30 seconds, Denature 94C for 30 seconds, Annealing 59C for 60 seconds, E 65C for 261 seconds. This cycle was repeated 30 times. Nanodrop readings were 502.5ng/ul for Sample 1 and 660 ng/ul for Sample 2. 

We then ran multiple gels on these reactions, with no real results to follow.