PCR Mix

 Primers

    We started off this week's research with a new project. We will be trying to create pigment-less Deinococcus (no orange). There are three sections we were focused on working with; left flank, right flank, and kanamycin resistance. To start the process we first needed to add primers to our solutions. We made a 20 micro-liter sample from 1 micro-liter forward primer, 1 micro-liter reverse primer, 2 micro-liter DNA sample, and 16 micro-liters of the master mix. After the solution was put together, it was placed inside the Thermal Cycler. The total process took over an hour and a half and went through 36 cycles total.

 


    The 36 cycles consisted of 7 different steps.
1. 95 degrees celsius (3 minutes)
2. 95 degrees celsius (10 seconds)
3. 65 degrees celsius (30 seconds)
4. 72 degrees celsius (1 minute)
5. Goes back to step 2 (repeat 35 times)
6. 72 degrees celsius (7 minutes)
7. 4 degrees celsius (until we are ready to remove)

    After we completed the Thermal Cycler process, it was time to nano-drop. For the Kanamycin we got 
192.8 ng/micro-liters, 1.82 A260/A280, 2.01 A260/A230
1.88 230, 3.94 260, 2.24 280

Gel Electrophoresis
    After we finished the nano-dropping, it was time to run gels. We had our 2 samples which included 18 micro-liters of our KAN sample, and then 2 micro-liters of our dye (we used 2 micro-liters of sample for the nano-drop). This time I made sure not to poke a hole in the bottom of my gel. Unfortunately, I ran the gel too long and the ladder fell off the edge. Luckily for me, we were going to remove the DNA fragment from the gel so we did not necessarily need the ladder. 


    Once the gel had finished running, we brought the gel over UV light and carefully removed the DNA fragment for future testing. 


    After the removal of our DNA, we incubated at 50 degrees celsius for 10 minutes (wanting the gel to completely dissolve). Through a process of adding different buffers and then filtering, we were eventually able to elute our DNA (extracting our DNA by washing it with solvents).

    
Once we eluted our DNA, we placed it inside the -20 degrees celsius freezer for storage until next week. 

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