High Molecular Weight DNA Extraction of Porites asteroides with the Qiagen Genomic Tip for Intended Genome Sequencing

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

This is from the successful attempt at extracting, the day before there was an unsuccessful attempt

Sample Chiseling

Set up

• Prepared buffer with 9.5mL of Buffer G2 and 19ul of RNase A
• Set incubator genie to 50 degrees C
• Cleaned chisel, forceps, scraper/scooper and bench 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 and LN2 in the Thermoflask dewar
• Brought over scale
• Set up dry ice box with plastic box rack over the ice to have a hard surface for chiseling, then covered that in foil

Chiseling

• Amy Zyck helped by holding the nubbin (literally unable to chisel without someone holding it)
• Took out nubbin: TA HOT 1 and unwrapped from foil (sample was flash frozen and kept at -80)
• Chiseled with large chisel and hammer until a few chunks came off
• Put mortar on the scale and tarred
• Used forceps to put chunks into mortar: 0.11g
• Decided to do a few more chunks (kit recommends 100mg, but hard to say how much skeleton I’m getting and how much will get scrapped out of the mortar)
• While chiseling again added LN2 to mortar to keep original chunks chilled
• Final weight 0.18g
• Poured LN2 into mortar and let boil off
• Ground chunks with pestle until powdery
• Scrapped into a chilled 50mL conical with the spatula
• Poured in the buffer G2 mix
• Vortexed briefly and placed in incubator genie 10 speed

Noticed ~45 min in that I forgot to add the pro K! Took conical out of the incubator and also noticed that it seemed to be leaking! There was liquid in the incubator, a few mL gone? Quick poured liquid into a new conical and added 500ul of pro k, vortexed and put back into the incubator for another 1.5 hours. This is longer than in the protocol but I was worried about there not being enough time to digest.

Genomic Tip Extraction

Genomic Tip

• Set centrifuge to 4 degrees C
• After incubation, transferred 1mL into each of 8 1.5mL tubes with wide bore pipette tips (some liquid had been lost)
• Centrifuged at 4 degrees C for 10 minutes at 5000 rcf
• Set up tip (resin column) inside a holder over a 50mL conical
• While that was going, added 4mL of buffer to the tip and let drip through to the conical (took the 10 min)
• After centrifugation, added the supernatant from the sample tubes (there was a small brown pellet in each tube) to the tip with wide bore pipette tip
• Started at 11:18 and was finished dripping at 11:31!
• Changed 50mL waste conicals
• Added 7.5mL of buffer QC (wash) and let drip through (10min)
• Warmed 5mL of buffer QF in incubator genie to 50 degrees C
• Repeated wash addition
• Transferred to a different 50mL conical
• Added the 5mL of warmed buffer QF and let drip through

Isopropanol Precipitation of DNA

• Made 6 1.5mL tubes each with 833ul of the elution liquid
• Added 583ul (0.7 volumes) of room temp 100% isopropanol to each tube
• Gently inverted to mix
• Centrifuged at 10,000 rcf for 30 minutes at 1 degree C
• Made fresh 70% EtOH and placed in -20 freezer
• Once finished, looked for pellets. Couldn’t see any, but could be glassy/clear
• One tube at a time, removed supernatant, leaving a little to protect potential pellets
• One tube at a time, added 1mL of cold 70% EtOH and vortexed briefly
• Centrifuged for 10 minutes at 4 degrees C 10,000 rcf
• Removed all of supernatant when finished. This time there was a small tan-ish supernatant visible
• Let tubes air dry ~7 minutes
• Added 100ul of TE bufer to each of the 6 tubes very gently
• Incubated for 1hr at 55 degrees C in the Theremomixer
• Once done, transferred to a shaker overnight 300rpm

QC

• Broad Range Qubit next day
• Flicked tube and took from both top T and bottom B

Standard 1	Standard 2	Sample	Average DNA ng/µl
151 RFU	21281 RFU	1 T	6.6
-	-	1 B	6.56
-	-	2 T	6.28
-	-	2 B	6.54
-	-	3 T	6.5
-	-	3 B	7.1
-	-	4 T	6.65
-	-	4 B	7.4
-	-	5 T	6.5
-	-	5 B	6.5
-	-	6 T	too low
-	-	6 B	too low

Well, the yield is kind of low, or at least not what I was hoping for. If say they’re 6ng/ul and you multiply that by 97ul and 5 tubes, that’s about 3ug of DNA. Not the 25ug we need.

What about the quality?

Genomic DNA screentape

Full Results

Amazing quality! Just have to extract more!