High quality DNA from Fungi for long read sequencing e.g.
PacBio
Make lysis buffer by mixing buffer A+B+C (2.5:2.5:1 + 0.1%PVP final) and briefly heat to 64 °C.
Let cool to room temperature for use in 50mL Falcon tubes.All following steps are based on 17.5ml lysis buffer as starting volume.
add 10uL (10kU) RNAse T1 to lysis buffer.
Grind tissue/spores with liquid nitrogen in a mortar with sand, use 1g of sand per 100mg of starting material.
Grind for 2 mins in 4x 15 sec bursts adding liquid nitrogen after each 15 sec grinding burst.
Transfer powder to 50mL Falcon containing lysis buffer and RNAse, mix well by vortexing.
Incubate at RT for 30 mins mixing by inversion every 5 mins.
Add 200uL Proteinase K, incubate at RT for 30 mins mixing by inversion every 5 mins.
Cool on ice for 5 mins.
Add 3.5 mL (0.2 vol) of KAc 5M, mix by inversion, incubate on ice for max 5 mins.
Spin at 4oC and 5000g for 12 mins.
Transfer supernatant to fresh Falcon tube containing 17.5ml (1vol) (P/C/I) and mix by inversion for 2 mins.
Spin at 4 °C and 4000g for 10 mins.
Transfer supernatant (might be milky but do not worry) to fresh Falcon tube containing 17.5ml (1vol) P/C/I and mix by inversion for 2 mins.
Spin at 4 °C and 4000g for 10 mins.
Transfer supernatant (~17mL) to fresh Falcon tube and add 5uL RNAse T1.
Incubate for 20-30mins at RT.
Add 1.8mL (~0.1vol) NaAc and mix by inversion.
Add 18mL (~1vol) RT isopropanol and mix by inversion.
Incubate at RT for 5-10mins.
Spin at 4 °C and 10000g for 30 mins.
Carefully pipette off supernatant till about 1-2 mL left, DNA will form a mostly translucent to white film/pellet at the bottom of the tube.
Use 1mL pipette tip to transfer pellet and remaining liquid into fresh 1.7mL eppendorf tube.
If the pellet got loose during transfer add 1.5mL fresh 70% EtOH to the 50mL Faclon and spin for 5min at 4000g.
Remove 1mL and transfer the remaining volume and DNA pellet to same 1.7mL eppendorf tube.
Spin in table top centrifuge for 5 mins at 13000g.
Remove supernatant with pipette and wash with 1.5mL fresh 70% Ethanol, invert several times to dislodge pellet.
Spin in table top centrifuge for 5 mins at 13000g.
Remove supernatant with pipette and wash with 1.5mL fresh 70% Ethanol, invert several times to dislodge pellet.
Spin in table top centrifuge for 5 mins at 13000g.
Remove supernatant with pipette.
Spin in table top centrifuge for 1 min at 13000g.
Remove remaining ethanol with pipette.
Air-dry pellet for 7 mins.
Add 200uL of 10mM Tris pH9 leave at RT for 3 hours.
Flick tube slightly for mixing and add 200uL of TE buffer.
DO NOT!
vortex as it shears DNA.
leave at RT over night.
Next day add another 100uL TE buffer and incubate for 1h at 28 °C with 1400rpm shaking.
Measure dsDNA concentration using BR Qubit and measure absorbance with Nanodrop.
At this point Qubit to Nanodrop ratios were 1/10 -1/20.This might be also a good step to assess DNA quality by runing a 0.8% TBE agarose gel with 500ng dsDNA and a lamda-Hind-III ladder as control.
If you have a Pulse Field Gel Electrophoresis around even better.
Use AMPure beads for secondary clean up at beads 0.45 (Vol/Vol) following the PacBio protocol.
Elute in 10mM Tris pH8.
Measure dsDNA concentration using BR Qubit and measure absorbance with Nanodrop.
At this stage Qubit to Nanodrop ratios were 0.64, 260/280 1.87 and 260/230 1.37.
Samples were submitted to Ramaciotti (http://www.ramaciotti.unsw.edu.au/) sequencing centre in Sydney.
Excellent personel performed quality control, prepared 15-20kb libraries and we ran 13 SMRT cells with P6 chemistry.
Some summary statistics are shown below.
