Second HMW DNA Extraction for Pac-Bio Sequencing of Pocillopora meandrina Flash Frozen Tissue Chunk

In this process I use the QIAGEN Genomic-tip 100/G, the QIAGEN Genomic DNA Buffer Set, QIAGEN RNase A (100mg/mL concentration), QIAGEN Proteinase K, and DNA lo-bind tubes

Goal: Get good quality HMW DNA from this sample, at least 6ug (to add to 19ug from the previous extraction)
Result: 28ug of DNA, with a little smearing
Major Take Aways: This is more than enough DNA, and the quality is really good, larger fragments than the first extraction, so I think we should use this extraction to send to sequence

2021 01 29

Set up

• Prepared digestion buffer with 9.5mL of Buffer G2 and 19ul of RNase A
• Set incubator genie to 50 degrees C
• Cleaned scraper/scooper with 10% bleach, DI water, then 70% EtOH
• Wrapped those in foil and placed in -80
• Also placed mortar and pestle in -80
• Had styrofoam cooler of dry ice saved from a shipment and filled Thermoflask dewar with LN2
• Brought over scale
• Set up dry ice box for keeping the conical and scraper chilled while grinding

Grinding and Incubation

• Picked out chunk with a good amount of tissue on it
• Tared the scale with the chilled mortar on it
• Put in the chunk and weighed: 1.55g
• Poured LN2 into mortar and let boil off
• Ground chunk until it was a powder. It was very hard to grind as the skeleton is very dense, I used LN2 two more times and really smashed the frozen tissue
• Scrapped into a chilled 50mL conical with the scraper
• Poured in the buffer G2 mix
• Vortexed briefly
• Added 500ul of Proteinase K and vortexed again, then spun down breifly
• Put the conical in the incubator genie at 50 degrees C rocking 15 speed for 2 hours

Genomic Tip Extraction

Genomic Tip

• Set centrifuge to 4 degrees C
• After incubation, transferred 1mL of lysed tissue liquid into each of 9 1.5mL tubes with wide bore pipette tips
• Centrifuged at 4 degrees C for 10 minutes at 5000 rcf to pellet any extra stuff
• Set up tip (resin column) inside a holder over a 50mL conical
• While that was going, added 4mL of equilibration buffer (QBT) to the tip and let drip through to the conical (took the 10 min)
• After centrifugation sample image, notice there was a small white pellet of debris in each tube, but not a lot
• Added the supernatant from the sample tubes to the resin tip with wide bore pipette tip and let drip through
• Dripping took ~16 min
• Changed 50mL waste conicals
• Added 7.5mL of buffer QC (wash) and let drip through (10min)
• Pipetted 5mL of buffer QF into a 5mL tube and warmed in incubator genie to 50 degrees C
• Repeated wash addition (10 minutes)
• Transferred resin tip to a different 50mL conical
• Added the 5mL of warmed buffer QF and let drip through (10 minutes)

Isopropanol Precipitation of DNA

• Made 6 DNA lo-bind 1.5mL tubes, 5 each with 833ul of the elution liquid, the last tube was about 680ul
• Added 583ul (0.7 volumes) of room temp 100% isopropanol to each of the first 5 tubes and 476ul to the 6th tube
• Gently inverted to mix
• Centrifuged all lo-bind tubes at 10,000 rcf for 30 minutes at 1 degree C
• Made fresh 70% EtOH and placed in -20 freezer to cool down
• Once finished, looked for pellets: there was a barely visible white pellet in each tube
• One tube at a time, removed most of the supernatant
• One tube at a time, added 1mL of cold 70% EtOH and vortexed briefly
• Centrifuged tubes for 10 minutes at 4 degrees C 10,000 rcf
• Removed all of supernatant when finished, used a p200 to get small volumes out, pellets were still visible
• Let tubes air dry ~7 minutes
• Added 50ul of TE buffer to each of the 6 tubes very gently
• Incubated for 1hr at 55 degrees C in the Theremomixer
• Once done, transferred to a shaker for 1 hour 300rpm
• Once done, tubes were placed in the fridge for the weekend

QC 2021 02 01

Qubit

• Broad Range Qubit the Monday morning (protocol)
• Tubes were taken out of the fridge and placed on the shaker at 300 rpm for ~30 min while the qubit standards came to room temp
• Flicked tube and took from both top T and bottom B of the tube measurements

Standard 1	Standard 2	Sample	Average DNA ng/µl	Averaged top and bottom ng/ul
179 RFU	19529 RFU	7 T	84.6
-	-	7 B	85.3	84.95
-	-	8 T	87.1
-	-	8 B	88.2	87.65
-	-	9 T	102.5
-	-	9 B	113.5	108
-	-	10 T	107
-	-	10 B	127.5	117.25
-	-	11 T	109.5
-	-	11 B	106.5	108
-	-	12 T	103.5
-	-	12 B	106.5	105

Genomic DNA TapeStation

• Followed Tapestation protocol
• Results link
• Most of the DNA is HMW, there is very minimal smearing, and the ladder worked perfectly

Total Amount of DNA from this extraction based on average top and bottom Qubit values and ~47ul of sample volume in each tube

• 7: 3992.65ng DNA
• 8: 4119.55ng DNA
• 9: 5076ng DNA
• 10: 5510.75ng DNA
• 11: 5076ng DNA
• 12: 4935ng DNA
• TOTAL: 28709.95ng DNA or 28.7ug of DNA