Used 1.4mm ceramic beads and the tissuelyser for 2 minutes at 25Hz, stopped at 30 seconds and moved the tubes to the other side of the sample holder in the lyser.
Incubated at room temp with the proteinase K for 15 minutes shaking at 550rpm
Sample Preparation
Samples:
“Tank Escapee” or TE (stored in DNA/RNA shield)
CCP1 - Chip - pentagona - ray (flash frozen)
BPH1 - Bash - hystera -ray (flash frozen)
Thawed TE on ice bucket
Prepared forceps, foil, and razor blades
Cleaned forceps with 10% bleach, DI water, 70% ethanol, and RNaseZap before and between each sample
Made 1 ceramic bead tube with 700ul DNA/RNA shield for each sample
Cut a small piece of tissue for TE then minced with the razor blade until it was flat and could come apart a little bit and placed in their respective bead homogenization tube
Cleaned forceps again for the FF samples, quickly took them out of the FF tubes and moved the tissue piece over into the ceramic bead tube with the shield
Ran the tissuelyser for 2 minutes at 25Hz
Spun down the bead tubes in the minifuge to remove bubbles
Added 70ul proteinase K digestion buffer and 35ul proteinase K to each bead tube
Briefly vortexed and spun down sample tubes
Placed in the thermomixer for 15 minutes shaking at 550rpm at 23 degrees C temp (room temp)
Removed all the supernatant (~700ul) into new 1.5mL tubes
Centrifuged tubes for 1 minute at 16,00rcf
Removed liquid (~700ul) into new 1.5mL tubes avoiding small pellet
Added equal volume (700ul) DNA/RNA lysis buffer to the 5mL tubes
Vortexed and spun down tubes
DNA Extraction
Warmed 10mM Tris HCl pH 8 in the thermomixer at 70 degrees C note did not warm H2O this time
Added 700ul of the liquid to yellow spin columns and collection tubes
Centrifuged 16,000rcf for 30 seconds
Saved the flow through in new 5mL tubes
Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (3 total spins)
Added 400ul DNA/RNA prep buffer to the yellow spin columns
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Added 700ul DNA/RNA wash buffer to the yellow spin columns
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Added 400ul DNA/RNA wash buffer to the yellow spin columns
Centrifuged 16,000rcf for 2 minutes
Transferred spin columns to new 1.5mL tubes for each sample labeled as final DNA tubes
Discarded flow through and collection tubes
Added 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
Incubated at room temp for 5 minutes
Centrifuged 16,000rcf for 30 seconds
Added another 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
Incubated at room temp for 5 minutes
Centrifuged 16,000rcf for 30 seconds
Discarded the spin columns
Made strip tubes with 7ul of each sample in them for QC
Kept tubes on ice bucket then stored in -20 degree freezer
RNA Extraction
Added equal volume (1450ul) 100% ethanol to the 5mL tubes of flowthrough
Vortexed and spun down
Added 700ul of the liquid to green spin columns and collection tubes
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (5 total spins)
Added 400ul DNA/RNA wash buffer to the green spin columns
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Created the DNase mix:
75ul DNA digestion buffer * 3 = 225ul
5ul DNase I * 3 = 15ul
Flicked and spun down mix
Added 80ul DNAse mix to each green spin column filter
Incubated for 15 minutes at room temp
Added 400ul DNA/RNA prep buffer to the green spin columns
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Added 700ul DNA/RNA wash buffer to the green spin columns
Centrifuged 16,000rcf for 30 seconds
Discarded flow through (Zymo kit waste)
Added 400ul DNA/RNA wash buffer to the green spin columns
Centrifuged 16,000rcf for 2 minutes
Transferred spin columns to new 1.5mL tubes for each sample labeled as final RNA tubes
Discarded flow through and collection tubes
Added 50ul room temp nuclease free water to the spin columns gently by dripping over the filter
Incubated at room temp for 5 minutes
Centrifuged 16,000rcf for 30 seconds
Added 30ul room temp nuclease free water to the spin columns gently by dripping over the filter
Incubated at room temp for 5 minutes
Centrifuged 16,000rcf for 30 seconds
Discarded the spin columns
Made strip tubes with 5ul of each sample in them for QC
Kept tubes on ice bucket then stored in -80 degree freezer
