https://padlet.com/brmontez/12kvs3makkve 11.17.2017 en-us 2017-11-17 21:16:05 UTC 2025-11-20 19:32:21 UTC hello@padlet.com brmontez https://padlet.com/brmontez/12kvs3makkve/wish/208306830 Unfortunately, due to our original GOI, REV-1, not properly being transformed into the plasmid back in week 7, we were not able to produce viable E. coli containing our GOI therefore not able to contribute to the rest of the experiment. For the duration of the lab, we worked with the original vector producing what is the control. The following results are of the gene Xpa-1, which was not tested by us, but rather is being utilized in this case for analysis. This is an analysis of what results should be expected.
- What is known about the gene are you investigating?   Your answer need not focus on these genes only in C. elegans; if similar genes exist in other organisms, you might describe the gene family/pathway broadly.
Xpa is....
- How did you manipulate the animals to perform the experiment?  (This is an introduction that would make, so you want an overview that works for your intended audience, not a highly detailed description.)

]]> 2017-11-17 21:17:24 UTC https://padlet.com/brmontez/12kvs3makkve/wish/208306830 brmontez https://padlet.com/brmontez/12kvs3makkve/wish/208307188
–pictures of GFP worms with/ without RNAi against GFP. 

]]> 2017-11-17 21:19:02 UTC https://padlet.com/brmontez/12kvs3makkve/wish/208307188 brmontez https://padlet.com/brmontez/12kvs3makkve/wish/208307419 Are there any conclusions that can be drawn from your results?

xpa-1 is required for normal survival and resistance to mutagenesis in response to UV light, the extended lifespan of dauer-like mutants, and fertility; XPA-1 is required in UV-irradiated nondauer larvae to prevent WWP-1-mediated proteolysis of AMA-1; transgenic xpa-1 rescues the UV sensitivity of the null allele xpa-1(mn157); xpa-1 is a core NER (Nuclear Excision Repair) factor (other members being xpf-1, xpg-1, and ercc-1); xpa-1 is essential for the repair of UV-induced DNA lesions and has been demonstrated to be required in both the global genome repair (GGR) and transcription coupled repair (TCR) pathways; following UV irradiation, XPA-1 activity is essential for normal meiotic development, induction of germ cell apoptosis, and the survival of germ cells and somatic tissue (even at relatively low doses of UV irradiation); loss ofxpa-1 activity via mutation or RNA-mediated interference (RNAi) renders animals hypersensitive to UV light at all stages of development; xpa-1 mRNA is detected in eggs and mixed stage populations.

]]> 2017-11-17 21:20:06 UTC https://padlet.com/brmontez/12kvs3makkve/wish/208307419 brmontez https://padlet.com/brmontez/12kvs3makkve/wish/208307712 We are testing if C. elegans have a difficulty repairing their DNA when the GOI (gene of interest) is knocked down by RNAi. We do this by exposing the worms to a mutagen, more specifically UV light. The worm strain we are working with, PD4251, carries a gene which allows the muscle nuclei of this microorganism to glow. If RNAi conditions are successful, the GFP (gene responsible for fluorescence in the muscle nuclei) should be reduced.
                                                                               
WEEK ONE
Creation of Media for RNAi Experiments
To a 500 mL flask, 293 ml of water was added.
An additional: 0.9 g NaCl, 0.75 g of peptone, and 4.5 g of agar were added. A stir bar was included in the mixture and sent to the autoclave for approximately 50 minutes.
(Due to the autoclave being utilized for another class, the remainder of the creation of media was completed for us.)

Moving of C. Elegans
8 adult PD4251 C. Elegans was transferred to two plates. 4 adults were transferred to one plate of a standard NGM seeded plate with OP50 E. coli, while the other 4 adults were transferred to a plate of NGM, IPTG, and AMP seeded plate with the GFP: RNAi bacteria. The progeny on these plates was observed to see if RNAi against GFP was successful.

Inoculated Cultures of Bacteria Containing RNAi Vectors
50 µl of 100 mg/ml Ampicillin and 125 µl of 5 mg/ ml Tetracycline was added to 50 ml of LB Broth. 5 ml of this mixture was transferred to individual tubes in which one tube was inoculated with GFP: RNAi bacteria and one tube inoculated with GOI: RNAi bacteria.

*Initiated Plates to Grow Adult Worms
A sterile loop was used to cut a 0.5 cm x 0.5 cm chunk of agar containing starved C. Elegans, and transfer it to an NGM plate containing OP50 strain E. coli. The NGM plate was incubated at room temperature for two days. to promote the growth of adult worms.

**Bacteria Cultures and L4/ Young Adult C. elegans Added to RNAi Plates
4 NGM, IPTG, AMP plates -referred to as RNAi plates- were inoculated via pipetting 100 µl of GFP: RNAi bacteria onto the center of two plates and 100 µl of GOI: RNAi bacteria into the center of the two remaining plates. 4 young adult worms were then transferred from the *initiated plates to each of the 4 RNAi plates, 2 plates with PD4251 worms on GFP: RNAi bacteria and 2 plates with PD4251 worms on GOI: RNAi bacteria.

Remove Adult Worms from the RNAi Plates
The adults from the RNAi plates were transferred to a new set of an additional 4 RNAi. Both sets of RNAi plates were incubated at 18° C.




                                                                                   WEEK TWO
**Exposure the Mutagen of UV Light
6 NGM plates were prepped, 3 labeled for GOI and 3 labeled for GFP. Each of the 6 plates received 4 young adult worms transferred from the RNAi plates. Each of the six plates was exposed to UV rays for either 0 seconds, 40 seconds, or 80 seconds. After exposure, the worms were then transferred to another set of plates and left for 24 hours to ensure the exposed adult worms laid eggs. One egg was laid, the adult worms we disposed of, and two days later the number of eggs and progeny were observed and recorded.

Observation of GFP in C. elegans
A thin chunk of agar was sliced and transferred to a microscope slide. 1 µl of 1 M Sodium Azide was added to the slide. A Luma Scope was utilized to observe the worms. ]]> 2017-11-17 21:21:29 UTC https://padlet.com/brmontez/12kvs3makkve/wish/208307712 brmontez https://padlet.com/brmontez/12kvs3makkve/wish/208396683 - the numbers of progeny on the “C” plates after exposure to UV (or not, for the controls). ]]> 2017-11-18 18:25:18 UTC https://padlet.com/brmontez/12kvs3makkve/wish/208396683