Catch up of July

I can't believe I am already on week 9. This blog will be extra long, because I have to fill you in on July 12-August 3.
Outside of the lab I have:

• Traveled to Fargo
• Ridden a ferris wheel inside the original Sheels sporting goods store.
• Been kidnapped, blind folded, and taken to the school planetarium to watch HP 7.1 on the big screen
• Watched HP 7.2
• Eaten at the locally famous "Red Pepper", Blue Moose, Little Bangkok, Rhombus Guys. As well as Happy Joe's and Applebees.
• Had a hall dance party with glow sticks, strobe light, and black light
• Built a fort
• Watched the sun rise from the top of the parking garage
• Played kickball
• Went swimming at a hotel pool
• Went star gazing
• Walked across the Mississippi river headwaters
• Bowling
• Sardines in Walmart
• Slack line
• 9 mile tandem bike ride at 11pm
• Baked a cake
• Cooked pancakes and bacon on my griddle

In lab:

• PCR: Creating the mutation on a template DNA plasmid
• Transformations: inserting the DNA into E. coli
• Culturing E. coli: Growing cells on petri dishes, picking colonies, and growing those colonies in flasks
• Minipreps out the wazoo: purifying DNA from E coli
• Restriction digests: cutting the DNA with special enzymes that recognize specific base pair sequences
• Gel electrophoresis: pushing the digested DNA through agarose to separate cut DNA pieces of different lengths (This is a check to make sure we have the right DNA) We can view the bands of DNA under a UV light.

• Sequencing the DNA: a long complicated processes that I don't really understand, but it gives me the base pair sequence of the DNA to make sure the correct mutation was created and no additional ones were made. Each wave in the chromatogram pictured below represents a different base pair.

• Culturing HeLa cells: Taking care of the immortal cell line of HeLa cells. Passing them into new flasks every 3 days to keep them from getting over crowded, and giving them new food to eat. 

• Plating HeLa cells: Counting my cells and putting 50,000 into each well of a 24 well plate.
• Transfecting my mutated DNA into my HeLa cells: Inserting the mutated DNA into the HeLa cells on the plates.
• Uptake of radioactive serotonin with a serial dilution of inhibitor: The DNA that was inserted into the HeLa cells sits over night and the cells will read the DNA and start creating serotonin transporters in their cell membrane. I then add inhibitor (citalopram-an antidepressant drug generically referred to as Lexapro) at various concentrations. Then I add radio active serotonin, which should be transported into the cell through the newly created transporters. 
• Counting radioactivity inside the cells: The 24 well plates are then put in a machine called a Top Counter, which counts the amounts of radioactivity inside the cell, which tells us how well the transporter worked or didn't work. 
• Creating a competition curve from the Top Counter data: A graph of the results will show a curved line on the axies of inhibitor concentration and radioactive serotonin uptake. The curve will shift left or right depending of if the mutation in the transporter is helping or hindering the uptake. If it is being hindered, then we can assume that the place where the mutation was created is most likely a binding site of the inhibitor.

It has all been really exciting to watch my project get to the results stage. I won't have time to run the experiment on all of my created mutations, but someone else in the lab will continue my project after I leave.

I am to the point where I feel accomplished, and I can leave feeling successful. I have learned sooo many lab techniques and computer programs. I have gotten to know my lab mates, and fellow REU students really well, and I am going to miss all of them.