Will Mullen

Introduction:

In order to be able run electrophoresis on the DNA that we had previously extracted using the Chelex method, PCR must first be done on the DNA. PCR stands for polymerase chain reaction which adds primers to the DNA in order to amplify it and allow it to be seen in electrophoresis. Two primers were used, the COX 1 primer and the Euk primer. Using two different primers will give us a better chance of seeing base pairs during electrophoresis.

Method:

1. The first step I had to take was to lower the concentration of DNA which was originally 230.43
2. I diluted it down to 23.043/100 ng
3. I did this by extracting 2.5 microliters of DNA and adding it to 10 microliters of water
4. First the amount of DNA, primers, and D.I. water were calculated in order to total the desired amount of 25 uL
5. Four tubes of PCR product were made
6. One tube had a control amount of the Euk and one was a control of the Cox1
7. The other two had either DNA and Euk primer or DNA and Cox1 primer
8. The tubes that contained the Euk primer were then Denatured at 95 degrees celsius
9. They were then annealed at 56 degrees celsius
10. They were then elongated at 65 degrees celsius
11. The tubes containing the Cox1 primers were denatured at 94 degrees Celsius
12. They were then annealed at 55 degrees Celsius
13. They were then elongated at 72 degrees Celsius
14. 35 cycles of the previous three steps occurred in order to get the most amplified DNA
15. They were then extended for 8 min at 72 degrees celsius for 8 minutes
16. Each tube was labeled I 1-4 in order to keep track of what was in each tube

Results:

• The final product was four microfuge tubes, two with a combination of primer, DNA, and DI water and two controls with no DNA. The tubes were stored at 4 degrees Celsius until the next lab experiment. The products in these tubes will be used to run electrophoresis in the next lab experiment. The controls will give a good contrast to the products containing the DNA because there will be no imaging on the controls.

Further Research:

This video really helped me to understand PCR better when we were actually about to perform it in lab. I found it to be slightly more beneficial than the one given to us mainly because of the animations and simple explanations. I really think that this video would help future students to understand the fundamentals of a truly fascinating process instead of just following the steps given to them.