Banana Slug Genome Project
A. Dissection
1. Received frozen slug, thawed in 37°C water bath
2. Made careful incision on side of slug, being careful not to break intestinal
3. Isolated and identified various body parts
4. Took out pieces of all major organs/body parts and put them into
5. Stored at -80°C
B. Extraction (using Omega E.Z.N.A. Mollusk DNA kit)
1. Took pieces of the digestive gland, albumen gland and kidney and
2. Placed a marble mortar and pestle into a bed of dry ice
3. Poured liquid nitrogen into mortar and placed sample into it, letting it
4. Ground sample to fine powder as the liquid nitrogen boiled off
5. Since the mortar is already cold, sample will remain in powder form even
6. Scooped powdered sample into tube containing buffer ML1 and
tissue
separate tubes
weighed them (25mg kidney, 120mg albumen, 60mg digestive gland)
freeze completely
7. Added chloroform:isoamyl alcohol (24:1), centrifuged at 10,000 x g for 2
8. We carefully pipetted the aqueous phase into a clean 1.5mL
when all of the liquid nitrogen boils off
Proteinase K, and incubated at 60°C until the sample was fully solubilized
(Note: albumen and digestive gland were split into two tubes/columns
due to mass of sample)
minutes. This step removes the mucopolysaccharides from our sample,
and leaves an upper aqueous phase containing our soluble DNA.
microcentrifuge tube and added buffer MBL and RNase A, and incubated
at 70°C for 10 minutes.
9. Once the sample cooled to room temperature we added absolute ethanol
10. Transferred 750ul of each sample to the HiBind® DNA Mini Columns
11. Spun columns at 10,000 x g for 1 minute, discarded filtrate
12. Repeated steps 10 and 11 until all of sample was applied to columns
13. Added 500ul HBC buffer with isopropanol, spun at 10,000 x g for 30sec,
14. Added 700ul DNA Wash Buffer with ethanol, spun at 10,000 x g for 1
15. Repeated steps 13 and 14 once
16. Dry spun for 2 minutes at maximum speed (14,000 x g)
17. Transferred columns to clean 1.5ul microcentrifuge tubes
18. Added 100uL Elution Buffer (preheated to 70°C), let sit at room
19. Stored DNA at 4°C
discarded filtrate
minute, discarded filtrate
temperature for 2 minutes, spun at 10,000 x g for 1 minute
C. Illumina Library Prep
(Two library preps side by side in two different size ranges)
1. Sheared DNA to two different sizes, ~400bp and ~600bp (6ug into each).
2. Size selected for two ranges, 350-500bp and 500-700bp.
3. Blunt End Repair with 50ul of sample plus the following:
Incubated at 25°C for 15 minutes then at 12°C for 5 minutes
Cleaned up reaction with 1.8x 18% PEG SPRI beads
Washed 2x with 80% EtOH
Eluted DNA in 20ul TE
Size selected, blunt ended DNA amounts:
350-500bp: 62.6ng/ul x 20ul = 1,252ng
500-700bp: 20.0ng/ul x 20ul = 400ng
4. Adapter Ligation with 20ul sample plus the following:
Incubated at 22°C for 30 minutes
Cleaned up reaction with 1.8x 18% PEG SPRI beads (same as last time)
5. Adapter fill-in with 20ul sample plus the following:
Incubated at 37°C for 20 minutes
Cleaned up reaction with 1.5x 18% PEG SPRI beads
Washed 2x with 80% EtOH
Eluted DNA in 30ul TE
Measure concentration by Qubit
300-550bp: 28ng/ul x 30ul = 840ng
550-750bp: 11.8ng/ul x 30ul = 354ng
6. Indexing PCR (index 89 for 300-550bp, index 90 for 550-750bp)
Performed 10 cycles of PCR with 200ng of each sample
(2 rxns each with 100ng each)
Thermal Cycler Parameters
1. 98°C for 3 minutes
2. 98°C for 20 seconds
3. 65°C for 30 seconds
4. 72°C for 40 seconds
5. Return to step 2 for 11 more times
6. 72°C for 3 minutes
7. 4°C forever
Measured by Qubit
350-500bp: 53.8ng/ul x 20ul = 1076ng
500-700bp: 60.4ng/ul x 20ul = 1208ng
7. Size selection of PCR product
Used E-gel SizeSelect 2% gel, selected for DNA in corresponding size ranges.
Measured by Qubit
350-500bp: 4.44ng/ul x 20ul = 88.8ng
500-700bp: 7.36ng/ul x 20ul = 147.2
D. Lucigen Nxseq Long Mate Pair Library Prep
Followed instructions exactly, with the exception of manually size selecting on
0.75% Agarose field inversion gel instead of selecting using AMPure beads