S-STEM Scholar Marisa Ostos Blog

Before I came into lab this week, my lab mentor went ahead and took my two tubes of inoculated E. coli with pRad1 (tubes "2.10A" & "2.10B") in LB + Ampicillin [50 ug/mL] out of the incubator and placed them into the fridge, this way, when I came into lab on Wednesday, I could go ahead and check the growth of my E. coli. After vortexing each tube and doing a nanodrop (OD600) reading, the following results were obtained:  (Figure A)  The above shows the two tubes ("2.10A" & "2.10B") that I did OD600 readings on, each of which showed strong growth of 2.28 & 2.20 for Tubes 2.10A & 2.10B respectively (ideally numbers of at least 1 are preferred) .  After this, I ran a plasmid extraction on each of the two tubes using the Zippy kit (Box B) ("ZymoPURE Plasmid Miniprep Kit" ). This was the same type of kit but a different box used (last week I used Box A). The results are shown below:  (Figure B)  These a...

After making mistakes last Wednesday with my plasmid extraction, I did go ahead and do nanodrop readings of my two sets of 2 tubes each (totaling four tubes). Here are the results:  (Figure A) The nanodrop (dsDNA) readings shown above represent each of the four microcentrifuge tubes shown below. In order, row 1 = "2.3A 1 of 2," "row 2 = "2.3A 2 of 2," row 3 = "2.3B 1 of 2," & row 4 = "2.3B 2 of 2." The tubes that were used to create these nanodrop readings are seen below :  (Figure B) These are the four tubes that were used to run the nanodrop readings seen, which correlate with the nanodrop readings .  Now, because I had forgotten to allow the elution buffer to sit for 2 minutes at room temperature before centrifuging, I did a "mini experiment" and re-eluted one of my four tubes (Tube 2.3B, which I felt was safest to experiment with as the nanodrop reading showed this tube as having the lowest concent...

This week's focus was on making sure growth was obtained with E. coli that has the pRad1 plasmid so that I could do extractions. It started out with the help of a lab-mate who, on Monday, was able to inoculate this particular E. coli from my plates into the 2X LB that I made last week (and since I had incorrectly inoculated E. coli with the pRadZ1 plasmid onto the plates initially, he inoculated these E. coli into two sets of 2XLB as well). On Tuesday, he transferred the four sets of E. coli from the 2X LB into regular [1X] LB (resulting in four total 50mL centrifuge tubes: two of which held E. coli with the pRad1 plasmid, and two of which held the E. coli with the pRadZ1 plasmid).  However, when I did a gram stain on each of the four LB tubes on Wednesday, I found contamination in all of them:  * From top to bottom are the clearly-contaminated E. Coli tubes with pRad1 (Tube A), pRad1 (Tube B), pRadZ1 (Tube A), and pRadZ1 (Tube B).  E. coli is gram negative and should be ...