Week of 4/22-4/26

 Introduction

    Having gathered all of the materials needed the prior week, our group was able to continue working on our Gibson Assembly project. Our Gibson Assembly requires three fragments, two of which we had prior to our temporary hold on the project. More of our left fragment was needed in order to carry on with assembly.

Methods

    On Monday we streaked 3 plates of D. rad, PRAD 1, and PHRAM using freezebacs. From these plates, the bacteria was then streaked onto other plates due to their coming from freezebacs. PCR was then conducted on our three fragments needed for Gibson Assembly. Next a gel was run to determine whether or not our three way Gibson Assembly was successful or not. Were it to be successful, the size of the three fragments combined would be 2,527 base pairs.

Results

    On Thursday we ran our gel to see if our 3 way Gibson Assembly was successful. An image of the gel is shown below: 

Gel Run on 4/25
Despite no band showing up on the 4th lane, a genomic extract control, on lane 2 a band formed at the desired 2500 base pair mark.

Conclusions

    No band being produced in the 4th lane was an oddity but it was mostly ignored due to the fact that we got a desired reading of 2500 base pairs on the 2nd lane. This would indicate that our 3 way Gibson Assembly was a success.

Comments

Post a Comment

Popular posts from this blog

Week of 10/28-11/1

This week, we inoculated 4-way E. coli into LB broth with 50 µg/mL ampicillin and performed a Gram stain on the growth, confirming it was free of contamination. The growth was then passaged into another LB broth with 50 µg/mL ampicillin for further experimentation. We also redid the NASA project run by inoculating D. rad. onto R2B and TGY broths. Additionally, we performed four plasmid extractions, but the nanodrop readings from these extractions were not favorable. Due to the less-than-ideal nanodrop values, these plasmid extractions were ultimately not used.
Read more

Week of 3/25-3/29: D. Metalli Biochemical Tests

Image
Introduction      This week we conducted biochemical tests on the bacteria D. Metalli in preparation for our conference presentation. These tests included the oxidase, urease, catalase, starch and SIM tests. These tests were conducted in order to better characterize the species.  Methods      In the beginning of the week we gram stained D. Metalli. Our results showed it to be a Gram positive rod shaped bacteria.  We then streaked our experimental plates, including R2A and TGY plates for catalase and oxidase tests, a Starch TGY plate for Starch hydrolysis tests, and 2 experimental Urease plates and SIM Test tubes for D. Metalli. In addition we also s treaked control plates/tubes with B. Subtilus, S. Epidermis, E. Coli, and C. Fruendii. The controls were incubated at 37℃ and the experimental groups at 30 ℃. The tests were then conducted on Wednesday. For the oxidase test, 2-3 drops of oxidase was put onto the plate. For the catalase test, drops of h...
Read more

Week of 9/16-9/20

This week, we poured 10 R2A plates and noted that the transformations performed on 9-12 and inoculated on 9-13 were unsuccessful. We tested Kanamycin’s efficiency on normal DRAD while also inoculating the most recent competent cells onto TGY with Kanamycin. To prepare for upcoming experiments, we planned additional plasmid extractions and made more media, including R2B and other types. We attempted to plate the previous transformation onto a TGY, Chloramphenicol, and Kanamycin plate and passaged 4-way E. coli into fresh LB ampicillin broth.
Read more