@bonesandbugs @UCSC_PGL I received samples from Jenny McGuire @JennyMcGPhD last week. They are small bones collected from Natural Trap Cave in Wyoming (#ntcave16 etc). This is the site where Julie Meachen @SaberCatWoman and her team (including me!) have been digging for the past five summers. 2/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman They've been sorting through all of the fossil bones, but some couldn't be identified by morphology alone. These include many broken bones (esp. of small vertebrates) & some ear bones (petrous or petrosals). Petrosals are hard & often are great sources for ancient DNA (aDNA). 3/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Here are two of the fifty petrosals that Jenny @JennyMcGPhD sent to me. Weigh boat is ~ 2 inches 4/x 
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman I spent most of the day on Thursday sorting through the samples, accessioning them into the lab & weighing them. FYI, those two weigh 105 mg & 236 mg, respectively 5/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Oh yeah, the bone fragments. I spent part of the day on Wednesday sorting through those and partially organizing them into rough size classes. We are putting those aside for the time being while we focus on the petrosals. More on that later. 6/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman On Friday, I cleaned 20 of the petrosals in preparation for aDNA analyses. It took me a few hours, since each has to be treated separately so as not to contaminate the others. Here's a snap of my lab notebook. 7/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman I convinced Katie Moon (postdoc in Beth's lab) to shoot some pics of the exciting process of cleaning the petrosals. Here I am removing the sample 8/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Here I am washing and surface cleaning the petrosal in 1% bleach. 9/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Next steps were to rinse with ultra pure water three times! 11/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Clean, but wet, petrosals were placed in caps (they were labelled!) to dry. 12/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Then it was time for cleanup! All of this was repeated for each sample (20 times). 13/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman The fruits of my cleaning were allowed to dry over the weekend. And I celebrated with a selfie. 14/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Today, along with master lab tech, Sam Cutler, we took 8 of these petrosals one step further...into bone powder! Sam took some pics while I powdered the 8th specimen. Step 1: prepare a clean space. 15/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 2: acquire sample and carefully wrap it in foil. Note use of extra mask. 16/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 3: smash the sample with Thor's hammer. 17/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 4: carefully unwrap the foil to reveal smashed up bone. This one didn't smash up very well, so we repeated the wrap and smash. 18/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 5: load smashed bone into heavy metal jar for use in shaker mill, add metal ball, tighten lid & load into shaker mill. 20/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 6: after 30 seconds of shaking, remove jar, open lid, and remove contents carefully (data not shown). Then weigh! 21/x
@bonesandbugs @UCSC_PGL @JennyMcGPhD @SaberCatWoman Step 7: transfer ~120mg of bone powder to fresh tube for DNA extraction. Any remaining powder is stored in another tube (I'm labelling that tube here). Next stop for these 8 samples will be DNA extraction on Wednesday morning! 22/x
Didn’t take any photos of DNA extraction & prep from yesterday & today. I’ll do that for future samples (there are 46 more!). But I’m very pleased to report that extractions were a success: the samples had > 8 ng DNA each. Plenty of DNA to move to the next step (libraries)! 23/x
Yesterday we prepared libraries from each of the first four petrosal samples (no pictures). Quantitative PCR indicates that the libraries are good! Next steps: library amplification & sequencing! 24/x
Today, I finished the DNA extractions from four more petrosals (5-8) that Sam & I started yesterday. We're checking the DNA concentrations now. Hopefully moving on to libraries tomorrow! 25/x
DNA concentrations for next four petrosals are all good to go for library making! Busy day ahead of us tomorrow! 26/x
Library making for petrosals 5 through 8 was delayed for 2 days due to power outages. However, I made them today! They are being tested using quantitative PCR, which also tells us how many PCR amplification cycles are needed to make the sequencing pools. 27/x
Speaking of which, the final library amplifications for petrosals 1 thru 4 are also happening this afternoon. Here they are. Sequencing happens on Monday! 28/x
Here are the data from q-PCR of petrosal libraries 5 thru 8. My libraries are on the left, negative controls on the right. Exponential amplification phase indicates Ct around 14 cycles for my samples! Good to move to amplification & sequencing on Monday! 29/x
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