Well, it’s been quite a while! The good news: it’s because I finally finished my analysis and have been focusing practically entirely on the writeup + future grant applications (which I, maybe wrongly, didn’t feel like was worthy of lab notebook writeups).
Anyway, I’m starting to figure out the feasibility of a large-scale Chionoecetes and Hematodinium sequencing project, using Pam’s old samples to track genetic changes over space and time. It seems like it could be extremely neat, and I’ll be writing things up in more detail later.
Of course, in order for any population genetics work to succeed, we need to successfully extract DNA from Pam’s samples! This might pose a challenge - they’re hemolymph samples stored in ethanol in capped deep-well plates, and many have been around since 2005. Some could be dried out or just unviable. So…Task 1 is Extract DNA from old samples!
I took 4 of Pam’s oldest samples (all from 2005) in a location where samples were extremely numerous (southeast Alaska). Here’s my plan to extract DNA and check quality. It’s largely (read: practically entirely) based on the Qiagen DNeasy Blood + Tissue kit instructions, as that’s what I’ll be using. Will chat with Steven soon to see how feasible it is!
Take whole sample (maybe ~200 ul), pipet into microcentrifuge tube.
Add 200 ul Buffer AL. Mix thoroughly by vortexing.
Add 200 ul ethanol (96-100%). Mix thoroughly by vortexing. Note: Is this step necessary? Already have hemolymph samples suspended in ethanol
Pipet into DNeasy Mini spin column in 2ml collection tube. Centrifuge at 8000 RPM for 1 min. Discard flow-through and collection tube.
Place spin column into new 2ml collection tube, add 500 ul Buffer AW1. Centrifuge for 1 min at 8000 RPM. Discard flow-through and collection tube.
Place spin column in new 2ml collection tube, add 500 ul Buffer AW2. Centrifuge for 3 min at 14,000 RPM. Discard flow-through and collection tube.
Transfer spin column to new 1.5ml or 2ml microcentrifuge tube
Elute DNA by adding 200ul Buffer AE to center of spin column membrane. Incubate 1 min at room temp. Centrifuge for 1 min at 8000 RPM.
Check DNA yield by running on BioAnalyzer.
If we see 10,000+ bp, that’s a really good sign. If we see 1000 or less, we’re in trouble.
Current big question: how do I actually go about running samples on the BioAnalyzer?
Alright, that’s it for now - meeting with Steven soon, should be able to figure out more details!
Misc notes from Steven meeting:
Might be good to spin down samples to pellet first, treat them as cultured cells. Can try in a few different ways.
Use heat blocks as incubators
Can also run on Qubit. Probably run on Qubit first, then run on BioAnalyzer
Store eluted DNA in -20, can QC it later.
Probably worth posting on GitHub asking for Qubit or BioAnalyzer protocol
Unsure if we have reagents for BioAnalyzer and Qubit