24 March 2014
ddRADseq, Phusion PCR, and gut microbesNext post Previous post
Awesome open science initiative. Would make a great teaching opportunity.
In general, this is a pretty cool open science platform: Synbiota
From Twitter, John Edmonstone, a former black slave that taught taxidermy to Charles Darwin!
Jake Russell: Microbial profiling of gut symbionts of ants
My question: how are the mutualisms (assuming actually mutualistic) maintained? In legume - rhizobia and fig - fig wasps, sanctions (or partner fidelity) can occur on ‘organ’ level. How is this done for the whole gut? Jake suggested that it might occur at colony level. That is…‘cheater’ strains will be weeded out through expression in individuals…does this make sense? Need to think about and/or model it…
Also pointed out in talk that can use ‘blank’ sample in Illumina sequencing to quantify contamination. Doh! Should definitely do this for RADseq.
The final stretch!
PCR for each size selected band (200, 300, 400) for each library (JSG001, JSG002).
Diluted stock PCR primers 2x to get 10uM working stock solutions.
Shared P1 for both libraries.
Used all 10ul of Dynabead-purified sample in PCR, as according to Dynabeads FAQ can use straight in PCR
For each sample, 20 ul reaction volume
|DNA Polymeras||e 0.2|
Made a seperate master mix for each PCR2 primer (one for each library). Ran PCR
|Final extension||1||72||5 min|
With the annealing temp from Yihong’s protocol with Pogos.
AMPure purification of the PCR-amplified product.
Eluted in 35ul.
Sent 2ul for Bioanalyzer, and will run a sample on Qubit tomorrow.
Gustafson, D.J., Major, C., Jones, D., Synovec, J., Baer, S.G. & Gibson, D.J. (2014). Genetic Sorting of Subordinate Species in Grassland Modulated by Intraspecific Variation in Dominant Species. PLoS ONE, 9, e91511.
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