Sep 8th (Prof Stephane)
@Member responsible for this page
Questions
- House keeping gene of amphibians
- general house keeping gene for all amphibians?
- I found a gene (called EF1-α: eukaryotic translation elongation factor 1 alpha) that is used as a reference gene for African clawed frog by a few papers and suggested to be a housekeeping gene for amphibians for one paper
- When I searched this gene on ncbi website, this gene was only appeared on African and tropical clawed frog among amphibians (this gene is present in many other eukaryotes). Is this because this is only sequenced on those?
- I know that the housekeeping gene are highly conserved, but yet its sequence may not be exactly identical from my understanding. So I am unsure whether this gene of African clawed frog could be used as reference gene for other amphibian species (especially when the sequence of other species couldn’t be found) Is it legitimate to use this gene as housekeeping gene for general amphibians?
- Also, I also when I searched for PCR assays on housekeeping gene, the genes are considered to be RNA (mRNA for most cases); it is converted to cDNA before amplification. Why do they choose this way? In considering the gene fragment that we are planning to amplify, is it okay to choose genomic one, not mRNA?
2. Approval process of using fungal samples in lab
3. We are currently in the works of deciding what DNA extraction/lysis method we are doing for our target fungi. Do you have any thoughts/expertise on DNA extraction/lysis methods that may work best on a fungal cell?
4. It is highly likely that we won't be getting our hands on the actual fungal sample that we are targeting for the iGEM project, so our plan is to purchase a fungal strain from a supplier that is most similar to our target. What type of cell characteristics do you think we should keep in mind when taking this decision, considering that we are using this fungal strain mainly to test our DNA extraction/lysis methods.
Meeting notes
- Positive control: you want gene to amplify in whole species ... why not use 16...mitochondrial gene?
16s mitochondrial gene - there are primers that work across all frogs
Find 16s mit gene and use predestined primers (plenty exist for PCR at least)
Can also use primers histone H3 (i searched it up, seems to be thats what its called)
- Most studies use reference gene's mRNA, not DNA for PCR/ nucleic acid detection.
- cheaper to get information about the transcriptome - expensive to sequence the population genomic information
- Cheaper to use mRNA (fill in)
DNA Extraction:
chitin cell well
Zymalase digestion .... not compatible with Bsal BD in literature
Justin thinks zymalase should work
In field, can boil with ..... to crack the fungal samples open (may cycle boil, freeze cycle too)
chelex
For In the field, try chelex - pretty dirty but it should work OR boil, cool cycles (recommended, followed by chelating agent after)
Potential fungal substitutes
Cell wall composition, cell size
Chtitrid fungus - could culture from soil if unavailable to buy Or contact people in the US to do simple stuff and ship the outcome
Could contact ——— Cornell and Anne Harbor.
https://ecologyandevolution.cornell.edu/kelly-zamudio
https://www.biorxiv.org/content/biorxiv/early/2020/07/02/2020.07.02.184721.full.pdf
Approval process for using fungal samples in the lab
Don’t consider anything that could cause disease, pathogenic
Anything close to pathogenic will take ages to get UAE approval
Sandra - if we grow fungi which has chitrid in it .... shouldn’t be a problem
think about what we can reuse the portable device
Skin swab can be tricky as not much DNA - SB unsure though
SG - salamander paper used inside cheek swab . Volunteers to catch and swab frogs for us
JW - frozen swabs from Ethiopia he can give us. In ethanol
Local frogs don't have chytrid
SB - prepare slides for what our project is, what we want, etc and present it to them. Steps for them to see our approach, so they can help us the best as possible. Cover everything, not just the biology side. Reps can go and present just a few slides
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