Team:BostonU/Notebook

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Day by Day

Protocols

Notebook

May

Week of May 18

• Made broth and agar. • Plated the agar. • Allocated media into 50mL portions to avoid contamination of an entire suppply • Innoculated protein plasmids from frozen stock. • Created cell stocks from frozen plasmids. • Miniprepped the cell stocks to isolate the desired plasmids • Used the nanodrop flourospectrometer to determine the concentrations of our plasmids

Week of May 25

• Digested the backbone plasmids were miniprepped • Set up PCR reactions to create split DNA sequences of the proteins we'll be using • Made an agarose gel and ran gel electrophoresis on the PCR results • Purified results from gel • Also created new cell stocks of plasmids that had low yields when originally minprepped • Miniprepped the new cell stocks • Measured concentrations of DNA from purified gel and miniprep • Digested backbones from miniprep in order to yield sticky ends for ligation of protein split into the plasmid • Gel electrophoresis from PCR reactions yielded strange results so we performed the PCR reactions again • Ran gel with new PCR results • Performed gel purification to isolate DNA • Digested and purified our inserts to complement the sticky ends we created in our backbones • Ligated our inserts into the backbones and transformed them into top10 cells • Plated the transformed cells to grow colonies • Checked the colonies after they incubated overnight, a couple had low yields • Picked three colonies of each ligation and placed them in deepwells to incubate overnight • Removed the deepwells from incubator, spun them down in the centrifuge and removed the media. • Miniprepped the cells from each deepwell to isolate the plasmid that contains both the insert and backbone.  • Digested a small amount of DNA from each ligation to perform a test cut. After digestion we ran the DNA through a gel. The desired result was two clear bands, one that was the size of the backbone plasmid and one was the insert. The actual results were not ideal so we decided to retrace our steps.

June

Week of June 1

• Picked new colonies from ligations that failed to show promising test cuts. • We also performed new PCR reactions on inserts that didn't look promising initially. We changed the settings on the thermocycler and also adjusted the amount of primers we were using per reaction. • Ran a new gel to see the results of the PCR reaction • Performed gel purification of inserts that were the proper length • We performed a new set of ligations which failed, since no colonies grew after the top10 cells containing the ligated plasmid were plated.

Week of June 8

Week of June 15

Week of June 22

Week of June 29