S-STEM Scholar Alex Hugenberg-Cox Blog

  After last weeks ligation failure and being unable to find a new ligation procedure, its been decided to put the restriction enzyme ligation project on pause. For now I'm helping out the overlap group again, which really just means helping Lani with whatever project she's currently doing.  While Monday was spent trying to figure out the restriction enzyme project, Wednesday I helped with a gel extraction and Friday we spent doing C1V1 calculations. Gel Extraction Procedures-       We started by removing the DNA fragment from the gel, putting it in a microfuge tube and weighing   it.     A fter we added the gel dissolving buffer, with 400 microliters of buffer per 100 milligrams of the gel     W e then incubated the sample at 50 degrees Celsius until it was dissolved, inverting it slightly every so often     T hen we inserted the column into a collection tube and loaded the sample. After spinning for a minute we discarded the flow through     W e then reinserted the co

After completing digestion and phosphorylation last week, we moved onto ligation. We would have started with measuring our samples on the fluorometer, however the machines readings were to low and inconsistent.  Ligation Protocol-  First setting up the tubes by adding the various components, and using a molar ratio of 1:3 vector to insert for the DNA.     Left and Tet  Right and Tet  T4 DNA ligase buffer  2  μl   2  μl  Vector DNA  50 ng (0.020 pmol)   50 ng (0.020 pmol)  Insert DNA  37.5 ng (0.060 pmol)  37.5 ng (0.060 pmol)   PCR water  to 20  μl   to 20  μl  T4 DNA ligase  1  μl   1  μl Then pipette the reaction up and down to mix, before briefly microfuging.  For sticky ends we then incubated at room temperature for 10 minutes, then heat inactivating both tubes at 65 °C for 10 minutes.  After ligasing

  This week of the linear plasmid project, we moved into doing the restriction enzyme digestion and phosphorylation. Before I started on the digestion protocols, I nanodropped the three successful pcr tubes from last week.  The tet sample measured at 513.4 ng/ μl . The two right samples measured at 369.8  ng/ μl  and 428.4  ng/ μl.  To begin with the digestion protocols, we first had to get 1  μg of each samples. Going off of our previous nanodrop measurements, we got 2.5  μl of left, 2.2  μl of tet, and 2.5  μl of right. After that, we set up each tube with its specific components.    Left Tet Right DNA 2.5  μl 2.5  μl  2.2  μl  10x rCutSmart Buffer 5  μl  5  μl  5  μl  BamHI-HF 1  μl 1  μl Xhol 1  μl  1  μl  PCR water 41.5 μl 40.5  μl 41.8  μl  Aft