S-STEM Scholar Aland Ali's Blog

 Introduction Last week in lab consisted of many plasmid isolations on Deinococcus Aquaticus. We've switched to using the ZymoPURE Plasmid Miniprep kit and have occurred many problems with concentrations since. Typically, readings of our plasmid ranged around 30ng/ul, but using this kit we've had concentration readings as low as 1ng/ul eluted in 25ul of elution buffer. We've also experienced problems during the neutralization stage of this kit. Normally, our solution should turn yellow with yellow precipitate almost instantaneously after inversion. However, our solution has stayed a pink color and formed some yellow precipitate. We predict that something in the resuspension stage is causing this issue. More specifically, we determined that our resuspension fluid is too diluted in our lysozyme, leading to cells not opening up properly. We are going to test this by having two samples straight from pellet to kit, and two other samples incubated in a lysozyme resuspension fluid...

Introduction This week in lab consisted of many plasmid isolations from various cultures of Deinococcus Aquaticus grown in TGY media. The concentrations of these samples have not been excellent, and genomic crossover is suspected. When observing a culture, precipitate was noticed and contamination had become a concern. Due to this, a gram stain was done on this culture.  Gram staining is a procedure used to test for infection or simply to stain bacteria. Bacteria can either be categorized as gram-positive or gram-negative in this test. After a series of specific stains, gram-positive cells will be stained red or pink; gram-negative cells will be stained blue or purple. These stains differentiate bacteria by the different aspects of their cell walls. For example, gram-postitive cells have many layers of peptidoglycan, whereas gram-negative cells have a much thinner layer of peptidoglycan; this makes certain stains only stick to gram-positive cells. Procedure To begin, a heat fixed ...

Introduction Earlier this week in lab, a 1% agarose gel was run on a plasmid from Deinococcus Aquaticus which we had grown last week. This gel had shown a 14kb band but none was the 7kb band we were looking for. We hypothesized that we needed to either incubate our sample in lysozyme for a longer period of time or use a different plasmid extraction kit in order to achieve better results. This was due to our sample not becoming clear after the addition of neutralization buffer from a GeneJET Plasmid Miniprep Kit. We will run another plasmid isolation using the same kit as a control in order to test this hypothesis. Procedure To prepare the sample for the ThermoScientific GeneJET Plasmid Miniprep Kit, I began by labeling 2 microcentrifuge tubes "A" and "B" respectively. I then tranferred 1mL of our TGY Deinococcus Aquaticus sample into each tube, and centrifuged for a minute. I carefully discarded supernatant and repeated this process 3 times for a total of 3mL in e...