Testing Charm DNA Kit on LCM Samples

Protocol Link

6/17/24 - Extracting DNA from LCM-captured cells (LCM date 6/13/24)

Used Charm Biotech Just-a-Tube ™ Laser Captured Microdissection (LCM) Sample Total DNA Purification Kit, protocol

Sectioning performed on 6/12/24 and LCM on 6/13/24. See details here

Protocol notes:

• Followed the Charm FFPE and Frozen tissue protocols almost exactly, but did all incubations at 56 ºC based on other Paxgene extraction protocols. Skipped the 94ºC incubation before the addition of ProK. Incubated in incubator genie (post pro K addition) for 2 hours. Then transferred lysate to new tube and spun down at 16000 rcf for one minute before moving supernatant into the supplied binding tube.
• Tube 10 was captured in 40 uL DD2 buffer (FFPE protocol)
• Tube 14 was captured in 40 uL DD1 buffer (Frozen Tissue protocol)
• Eluted in 15 uL of elution buffer and shook at 500 rpm for 5 minutes

Qubit Results

• Used High Sensitivity DNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 64.64 (S1) & 23642.49 (S2)

colony_id	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
LCM sample #10	0.701	0.697	0.699 ng/uL
LCM sample #14	0.209	0.206	0.208 ng/uL	<– I find this odd since there was a huge visible pellet for this tube during the extraction

Could have possibly overdried the pellets….

After heating the DNA to 56 ºC for 5 mins:

DNA Standards: 64.64 (S1) & 23642.49 (S2)

colony_id	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
LCM sample #10	0.804	0.792	0.798 ng/uL
LCM sample #14	0.577	0.571	0.574 ng/uL

Nice! Wonder how this would affect DNA quality on the tapestation. But a viable option to extract DNA, I think.

DNA Quality Check: Tapestation

Womp womp. Nothing. Maybe some smudges?

Full results can be found here