S-STEM Scholar Aland Ali's Blog

 Introduction Recently, a gel was run with various lysing methods performed with D. aquaticus. M23 primers revealed a brighter band with cells boiled for 15 minutes as a template, 7kb primers hadn't shown. A heat gradient will be performed on boiled cells to indicate the best range for 7kb primers to anneal. The range chosen for this heat gradient is 50-66 °C , additionally, initial denaturing will be extended from 30 seconds to 4 minutes to allow further separation of cells.  Additionally, alternate genomic DNA extraction of D. aquaticus will be used as a template for a long amplification PCR at 59 °C annealing. M23 (~700bp) and E. coli's crtb primers (~6650bp) will be used as controls for mastermix denaturation and long amplification on larger fragments. The reason for using culture run-through bead-beading is to determine the best template to use is boiled cells, as it was an unexpected outcome. The alternative DNA is D. aquaticus run through the DNeasy Ultraclean Microbial...

 Introduction  Recently, we had obtained E. coli's cm^r gene from pRAD1. However, when observing a gel for 7kb fragments, only primer bands were shown. Three hypotheses were that the master mix wasn't made correctly, the annealing temperature during PCR was too high for binding, and the primers could have been constructed incorrectly. To test these hypotheses, we'll run four long amplification PCR reactions. The two templates we'll use are boiled D.aquaticus cells and a plasmid extraction sample of D.aquaticus that was used when first constructing the current 7kb primers. Each pair will use m23 primers and 7kb primers. The purpose of the m23 primers is to be used as a control for master mix. The m23 primers are expected to prime to the m23 portion of D. aquaticus' 7kb plasmid and show on the agarose gel. We'll also lower our annealing temperature to 57 °C and expect more non-specific binding than from our 59 °C samples.  Procedure To begin making our 4 25ul long...

 Introduction  This semester, Leilani Boren, Micheal Dockham and I are transforming the 7kb plasmid from Deinococcus aquaticus with E.coli's chloramphenicol resistance gene via PCR amplification and Gibson assembly. Once assembled, the 7kb plasmid will be farmed and reintroduced into D. aquaticus. To do this, we must first prime D.aquaticus' 7kb plasmid as our vector and E. coli's chloramphenicol resistance gene as our antibiotic resistance gene via PCR.  DNA cloning works by inserting a target gene (cmr) into a plasmid (7kb) . Afterward, the plasmid is introduced into bacteria (E.coli) via transformation. Colonies will be screened via selective media (LB + chloramphenicol) and, once verified, will be used to farm recombinant plasmid DNA.  Typically, Plasmid DNA is manipulated via restriction enzymes which will cut at cloning sites to create an area for our fragment to be introduced and hybridized. However, we will be using the Gibson Assembly due to successes in com...

 Introduction Last week in lab, two largely concentrated samples of the 7kb fragment from Deinococcus Aquaticus was obtained. However, the gel showed this sample to be at 14kb. A theory for this is that two 7kb fragments are mineralized together. Therefore, we are using restriction enzyme to demineralize these fragments and have them appear at 7kb. If this isn't the case, we will use primers. Restriction enzymes are sequences that are recognized by restriction enzyme to cut plasmids. We cut at the restriction site xbaI (1188 base pairs) but this should show as 7kb on the gel.  Procedure A 1mL tube was filled with 68ul pcr water, 10ul cutsmart buffer, 2ul restriction enzyme , and 20ul of our plasmid isolation samples from the week prior (2mg). Once we have a 100ul total , we will incubate this tube on a heat block at 37 degrees celcius. Due to not knowing how long this enzyme will take to completely cut at all of the restriction sites, we will use 4 incubation time periods. The...

 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...