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Week of 11/18-11/22

This week, we checked the growth of transformation plates from the prior week, but no growth was observed. The control plates showed only pigmented growth, and the plates exposed to UV radiation on normal TGY also did not show any growth. Additionally, rehydrated normal D. rad. cells for the NASA project failed to grow. To address these issues, we planned and researched a new transformation protocol and successfully identified one specifically designed for D. rad. The protocol is as follows: 1. Take 5 mL of fresh D. rad. growth (broth) and transfer it into a 15 mL tube. 2. Add 1 mL of CaCl_2 to achieve a final concentration of 30 mM. 3. Incubate for 80 minutes at 30°C, then transfer 1 mL to a 1.5 mL tube. 4. After incubation, add 10 µL of plasmid and hold on ice for 30 minutes. 5. Dilute each tube with an additional 9 mL of TGY broth and incubate for 18 hours at 30°C. Using this new protocol, we performed three transformations. We also prepared one plate of normal TGY with two sampl...
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Week of 11/11-11/15

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This week, we rehydrated D. rad. growth for the NASA project and plated it onto respective plates for desiccation. A Gram stain of 3-way D. rad. from a plate confirmed it was free of contamination, and this culture was inoculated into a new TGY Kanamycin broth. We also passaged 4-way E. coli into fresh broth and performed three plasmid extractions from the culture. Successful growth of 3-way D. rad. in broth was verified by Gram stain, and a Gram stain of 4-way E. coli confirmed its growth in broth was also free of contamination. On 11/12, we conducted a gel on the plasmid extractions, which showed strong bands around 10,000 base pairs, as expected. Three transformations were performed, though contamination was found on one of the transformation plates. To address this, we re-inoculated the transformations and exposed them to 200 microjoules of UV for five minutes. Additionally, we conducted DNA extraction on 3-way D. rad. four times, with three of the trials yielding very favorable re...
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Week of 11/4-11/8

This week, we inoculated 3-way D. rad. from a plate into TGY broth with 8 µg/mL Kanamycin but continued to encounter difficulty with its growth in the broth. We passaged 4-way E. coli into a new LB ampicillin broth, but a Gram stain revealed signs of contamination. To address this, we inoculated new LB ampicillin broth with 4-way E. coli from a freezeback and prepared 250 mL of LB Agar. A Gram stain of the new 4-way E. coli growth confirmed it was free of contamination. For the NASA project, desiccated D. rad. was unable to grow, so we inoculated normal D. rad. into TGY broth and R2B for desiccation. A Gram stain of this D. rad. confirmed it was also free of contamination.
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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.
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Week of 10/21-10/25

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This week, transformations performed on 10/16 and plated on 10/17 were not successful. In preparation for a new run of transformations, we made two 250 mL batches of TGY Agar. Using these, we poured 10 TGY Chloramphenicol plates with a concentration of 3 µg/mL and 10 TGY Kanamycin plates with a concentration of 8 µg/mL. Additionally, we ran a gel on three plasmid extractions, but the results did not look favorable.
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Week of 10/14-10/18

This week, we conducted two Gram stains on potential 4-way D. rad. growth observed on a Chloramphenicol plate and passaged this growth onto a new Chloramphenicol plate. We prepared 250 mL of TGY Agar and poured 4 TGY Chloramphenicol plates with a concentration of 3 µg/mL and 5 regular TGY Agar plates. A Gram stain was performed on the new Chloramphenicol plate with potential 4-way D. rad. growth, and the growth was passaged again onto another new Chloramphenicol plate. However, a subsequent Gram stain on this most recent passage revealed that the transformation was unsuccessful and contaminated. We planned to perform new transformations and are considering exposing plates to UV light immediately before incubation to improve results.
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Week of 10/7-10/11

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This week, we poured 10 TGY plates with 8 µg/mL Kanamycin and inoculated 3-way D. rad. into 25 mL of TGY with 8 µg/mL Kanamycin broth. We performed three transformations using previous plasmid extractions, but unfortunately, the transformations did not succeed. To check for contamination or other issues, we Gram-stained 3-way D. rad. from older broth stored in the fridge. Additionally, we rehydrated desiccated D. rad. cells using a rehydration buffer. We also passaged 4-way E. coli into fresh broth and inoculated more normal D. rad. in preparation for the NASA project. Finally, we performed three additional plasmid extractions on 4-way E. coli. The nanodrop values for these extractions are included via image.
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