9/12/18

Continuing on with our experiment, I started with running the PCR products on gels. It was the same procedure as from the last week’s lab. I made array of 2µl dots of loading dye onto the Parafilm and made total of 12 dots for my lab group. And on top of the loading dye, I added 3µl of PCR product from the top to bottom: Kayla, Carter, Peter, and Jinwoo. After that I loaded the gel along with the latter and recorded the location of my PCR product.

After Gel electrophoresis was finished, we checked our genomic DNA and unfortunately, I couldn’t see my DNA on the gel. So… I made another master mix and using my gDNA, I created another PCR product so that Professor Paul can get my genomic DNA for DNA barcoding.

If my genomic DNA were to be shown on the gel, I would have done ExoSap PCR clean up.

Materials for ExoSap PCR clean-up:

-H2O

-10x buffer (Sap 10x)

-SAP

-Exo

-Ice

-Thermocycler

Procedure:

First, I was suppose to determine the number of PCR clean-ups that I have to do and calculate the volume of Master mix needed for my PCR. Then Pipette 7.5µl of each PCR product into a clean, labeled 0.2µl PCR tube and put it into a bucket of ice while making the ExoSap master mix. After making master mix, pipette 12.5µl of master mix into each PCR product tube (3 total if you had 3 genomic DNA found). Next, I would have placed the tubes in a thermocycler and start the ExoSap program. After the program is completed (~45 minutes) place PCR tubes in a labeled tube rack and place in the freezer so that it can get sent to somewhere that can do DNA barcoding.

p.s. I don’t know if my PCR product succeeded or not.