Objective and summary:

• Complete extraction and qubit quantification using non-target adult scallop samples

  • 30 mg and 90 mg of tissue
  • 3 hr Proteinase K incubation at 120 RPM and 55C - vortexed every hour
  • doubled the volume of TL Buffer, HBC buffer and 100% ethanol for the 60 mg samples (did not double proteinase K)
• DNA was extracted using the OMEGA EZNA Tissue kit protocol here
• Ran Qubit dsDNA BR kit following standard protocol thermosci online pdf

Qubit Results

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Sample	DNA (ng/uL)
	RUN 1	RUN 2
Standard 1 30 mg	77.46	76.85
Standard 2 30 mg	9084.45	8959.47
orange 1 MA 30 mg	19.0	19.0
orange 2 MA 30 mg	7.74	7.65
blueyellow MA 30 mg	5.12	5.14
greenwhite MA 30 mg	8.67	9.31
orange 1 MA 60 mg	85.3	83.7
orange 2 MA 60 mg	27.2	27.1
blueyellow MA 60 mg	7.57	7.69
greenwhite MA 60 mg	10.1	10.1

Moving forward…

• as we see here… the overnight 55C incubation in previous DNA extractions is the likley culprit!
• we also
• for te next extractions…

(1) Use more tissue

• the protocol (OMEGE EZNA) asks for 30 mg of tissue and I have been following this - double the tissue and proteinase K to see if this increases DNA

(2) Decrease volume of Elution Buffer

• use 2 elutions at 50 ul each as opposed to 100 ul. This should at least double our possible DNA concentration - our main bottleneck for library prep is concentration of DNA and not total DNA so highly concentrated low volume is perfect