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 .
