Ethanol precipitations

Today I did ethanol precipitations for samples 7, 11, U2, U3, and U6 following this protocol:

• I centrifuged the samples at room temperature
• After washing the pellet with 70% ethanol, I removed the supernatant before adding more 70% ethanol.
• I warmed the E.Z.N.A. Elution Buffer to 70ºC before adding it resuspend the pellet

I then ran the samples on the Qubit!

Table 1. Volume (µL) reagents added for ethanol precipitation and Qubit concentrations. S1 = 340.74, S2 = 37549.58

Sample	Treatment	Volume (µL)	NaOAC Added (µL)	100% Ice Cold EtOH Added (µL)	70% EtOH Wash Added (µL)	Concentration (ng/µL)
07-T2	Low	40	4	80	400	18.5
11-T4	Ambient	30	3	60	400	20.4
UK-02	Ambient	45	4.5	90	400	21.0
UK-03	Ambient	40	4	80	400	14.2
UK-06	Ambient	45	4.5	90	400	47.0

Samples 7 and U3 needed to be precipitated again, so I did just that before checking the concentration again.

Table 2. Volume (µL) reagents added for ethanol precipitation and Qubit concentrations. S1 = 159.38, S2 = 16594.26

Sample	Treatment	Volume (µL)	NaOAC Added (µL)	100% Ice Cold EtOH Added (µL)	70% EtOH Wash Added (µL)	Concentration (ng/µL)
07-T2	Low	20	2	40	300	22.4
UK-03	Ambient	20	2	40	300	11.4

Sample 7 was fine, but U3 had a lower concentration than it did previously! I started to get suspicious about DNA loss so I updated sample concentrations and volumes. I definitely lost a considerable amount of DNA and I did not save any of the supernatant…fuck. I posted this issue to get more information. I’m SOL for these samples unless I extract more DNA, but I wonder if there’s a low-input WGBS option. At least I have the ability to re-extract DNA for 7, 11, U2 and U3 if needed, but who knows if I can get RNA from them too.

Going forward

1. Figure out what to do about samples (either extract more or find a low-input WGBS option)
2. Prepare dilutions and run them on the BioAnalyzer
3. Extract RNA from gonad samples