Lab 8: PCR findings, gel electrophoresis and Group poster design

Adriana Robledo

3-8-19

Purpose:

The purpose of this lab was to perform gel electrophoresis as a way of measuring the success of the samples that underwent PCR in order to determine whether or not they should be sent to a lab for secondary analysis. The results and techniques from lab will be incorporated into our group posters which will be organized during the period.

Procedure:

1. Working with a 1.5% agarose gel pre-prepared by lab staff, label the order in which you will be placing your DNA in the wells. Record this order into your journal
2. Pipette 5μl of the 1 kb ladder for your first well followed by 10μl of DNA into their appropriate wells, careful to keep track of the order
3. Place the gel into the electrophoresis chamber (negative end with the wells will face the black) and attach the wires
4. Run the gels at 100V for about 30 minutes (Dr.Adair will analyze the gels in the meantime, and she will release the photos later in the day)
5. Head to the computer lab to take the midterm, then begin the poster outline with your group (following the group discussion)

Results:

Well order (gel 3):
1. ladder

2. MAC

3. MAC control

4. GD

5. GD control

6. CJ

7. CJ control

8. EMPTY

Conclusion:

Our protocol seemed to be a successful one seeing as a peer whose sample occupied well 6 (using the silica bead method) was able to get a great range for his DNA, but our sample, specifically, was not. Dr. Adair seemed to believe that our DNA was too concentrated (resulting in what looked like a “smear” of DNA, with no definitive bands) which may have resulted from an improper dilution. I don’t know if we are going to try the process again (which seems unlikely seeing as it took so long to get to where we are now) but I am open to the idea and maybe finding the specific point in which we went wrong. It would have been exciting to see that we were successful but this technique was very new and understudied so it is important to have these trials and errors. The midterm quiz was basically what we have been doing thus far which was a bit tricky in the end. We are working with very complicated techniques that are dependent on an understanding of the process by those using it. With that being said, the poster will allow us to display our knowledge that we have gained in this course (which was tested by the quiz) and organize it in a way that will help others see the techniques, and document them for future use and improvement upon.

Future steps:

This lab really was the deciding factor between continuation of sequencing or not. Out data was very concentrated so we may have to repeat the procedure in order to get a workable sample for further studies. The next step (is successful) would be to BLAST the samples and get an understanding of the diversity.

Storage:

Micropipettes were stored, and their used tips were discarded. The tubes of DNA that we used were returned to their ice.