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Monday, July 12, 2010

Our procedures...

In the Pritzker Laboratory scientists use different variations of procedures to carry out their research. Much of lab work is based off of preference. In the Pritzker Lab, although there are protocols, each individual manipulates the protocol to fit their project based on coherence and validity. A few of the major procedures that most of the scientists in the lab use are PCRs, gels, and sequencing; however, as mentioned before, each team or scientist tweaks the protocol. Thus, we will be showing you the methods we (Team Shark) use to carryout our research.

Lets Begin:
1. The first procedure we do is a PCR or polymerase chain reaction. A PCR is a way to amplify or make millions of copies of a specific gene sequence of DNA, making it much easier to study and analyze later. A PCR only requires a small amount of DNA, so it's very easy to make tons of copies to work with after starting with only a small amount of tissue or blood to extract from.

How to do a PCR:

*NOTE: We did this PCR with DNA that has already been extracted from a shark's fin.

2. The next step is to see if our PCR was successful. For these purposes we run a Hi-melt Agarose gel. We pipette dyed DNA into a gel and run it through an electric current. DNA fragments move through the gel from negative to positive (since DNA is a negatively charged particle). Shorter bands travel farther through the gel while longer bands travel shorter distances. Using ultraviolet light, we can see the DNA on the gel and see if the bands we are looking for appear. If the samples look good, then we're ready for sequencing.

How to run a Hi-melt Gel:

3. Finally, we determine banding patterns using fragment analysis. We add the finished and checked PCR samples to a master mix of LIZ500 which acts as a ladder for comparison and Hi-Di formamide which denatures DNA. We add the samples and master mix to a sequencing plate which is loaded into the 3730, a machine worth a quarter of a million dollars. Once the 3730 is done analyzing, we can pull the data off a computer and begin scoring the data.

That's the overall procedure and goal of Team Shark. It may sound simple (or extremely difficult) but we repeat these same protocols over and over again for many loci. When we run into errors, we sometimes have to repeat everything over again. Working in the Pritzker Lab has truly taught us the importance of patience.

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