Sam’s Notebook: MBD Enrichment – C.virginica Sheared Mantle DNA from 20190306

Last week I performed MBD enrichment on 12 of the 24 Lotterhos C.viriginica mantle DNA on samples I had sheared on 20190306. I proceeded with MBD enrichment using the MethylMiner Kit (Invitrogen) on the 12 remaining samples of the 24. I followed the manufacturer’s protocol for input DNA >1ug – 10ug with the following modifications:

• 2500ng of input DNA used
• discarded washes post-DNA-binding
• performed high-salt elution

The full sample list and calculations are here (Google Sheet):

• Roberts_2017AdultExposureSampleInfo

Will proceed with ethanol precipitations of all 24 samples.

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Homogenization and RNA treatment: round 2 of samples

3/4/19- 9:40 AM

Using samples 101, 103, 110, 116, 122, 128, 138, 141

I removed the eight samples from the -80 and immediately put them on dry ice. Once I began homogenizing the tissue using the pestle and mortar, I had some trouble getting all of the tissue homogenized for the first two samples (138, 122), so I did not keep half of the powder. 122 and 141 also fell out of the bowl of liquid N2 during grinding, so we’ll see what turns up with that. 116 was a very small sample, so I did not end up getting much powder into the buffer. This time, I did step 3 (DNA spin column) and kept the columns for 128, 138, 110, and 101.

Put everything (original sample tubes with half of powder, current samples with completed buffer, and DNA columns) into Box 3 in the -80. Will return to do treatment this week. 

3/7/19- 8:30 AM

I took the homogenized samples out of the -80 and began the treatment yo extract RNA. Spin columns clogged again, so I increased the speed to last week’s (full speed). I kept the spin columns from the last step (10), but did not keep the flow through.

Everything is in the -80 this time, in 2 boxes labeled with my name, “samples,” and date, under Laura’s samples. Next week I will quantify these and begin the third round of samples.