PURPOSE: the purpose of this lab was to explore PCR and prepare the gel for gel electrophoresis for next class. The purpose of PCR is to amplify a segment of DNA via the use of specific primers that bind at a specific area on the strand of DNA and then replicate it. We do this in order to have enough DNA to run gel electrophoresis and get results.

PROTOCOL:

PCR:

1. Review the QTM and determine the volumes needed for each of the tubes. We will have a positive control, a negative control, and an experimental tube.
2. To the positive control tube add 12.5 ul of 2X master mix, 5 ul of Paramecium DNA template, 1ul COX1 primers, and 6.5 ul of water to equal a total volume of 25ul.
3. To the negative control tube add 12.5 ul of 2X master mix, NO DNA TEMPLATE, 1ul COX1 primers, and 11.5 ul of water to equal a total volume of 25ul.
4. To the experimental tube add 12.5 ul of 2X master mix, 5 ul of extracted soil ciliate DNA template, 1ul COX1 primers, and 6.5 ul of water to equal a total volume of 25ul.

PCR Cycling conditions:

1. run at 94C for 4 mins
2. 5 cycles of denaturation at 94C for 30s, annealing at 45C for 1 min and extension at 72C for 105 sec
3. 35 cycles of denaturation at 94C for 30 s, annealing at 55C for 1 min and extension at 72C for 105 sec
4. Hold at 72C for 10 min

Gel Electrophoresis:

1. To make a 1.5% agarose gel, add .6g agrose to 40ml 1xTAE in an Erlenmeyer flask.
2. lightly cover the top of the flask with a piece of the weighing paper so that the solution does not boil over.
3. Put the flask in the microwave for 1:30 sec on 70 power or just until the solution begins to boil
4. Once the solution begins to boil, remove and put in an ice bath so that it is easier to handle
5. Once the solution and glass have cooled add 2 ul ethidium bromide into the solution and swirl
6. pour the solution into the mold and allow to solidify for 25-30 mins.
7. if not using immediately, store in the fridge

RESULTS:

sequence of forward primer: 5′-ATGTGAGTTGATTTTATAGAGCAGA-5′

sequence of reverse primer: 5′-GGDATACCRTTCATTTT-3′

Tube “P”: 25ul (DNA concentration 10ng/ul)

Tube “N”: 25ul

Tube “E”: 25ul (DNA concentration 164 ng/ul?????)

*concentration of primers in tubes= .4uM

Stored in rack: A10, A11, A12

Mass agrose: .6g

Volume 1xTAE: 4oml

CONCLUSION:

We still aren’t sure that this protocol is the best for soil ciliate especially since our personal nucleic acid concentrations weren’t originally good from the EZNA protocol. The next step is to run the gel electrophoresis and compare our data in order to identify the ciliates present. We must keep in mind that the positive control is going to have mainly all 1-2 types of paramecium where as ours most likely contains many different organisms which is why we used our specific primers however this might result in faulty results.