Isolation of high molecular weight genomic DNA from Entomophthora muscae

Fungal culture can be established from glycerol freezer stock or from a sporulating cadaver following methods outlined in Hajek et al, 2012. Cultures are grown in supplemented with added 5% FBS (e.g., ) , which will henceforth be referred to as Grace's + 5% FBS .

From -80C glycerol stock

Transfer 20mL of Grace's + 5% FBS to and allow to come to room temperature.

Retrieve one 1.5mL-2mL aliquot from -80°C and rapidly thaw entire aliquot at 37°C.

Transfer entire contents to prepare tissue culture flask.

From sporulating cadaver

Collect conidia via ascending method per Hajek et al, 2012. Briefly, using aseptic technique:

-Adhere cadaver via ventral abdomen to the top of an inverted sterile petri dish and allow to sporulate for several hours (at least 2h 0m 0s and no more than 15h 0m 0s) onto the base of the dish.

-Replace the top of the dish, invert the dish and cover spores with room temperature Grace's + 5% FBS.

-Incubate dish for at least 48h 0m 0s at room temperature, monitoring for contamination. If the dish becomes uniformly cloudy at any point, it has been contaminated by something other than E. muscae .

-Once clumpy, protoplastic E. muscae growth can be observed, transfer culture to 25 cm^2 flask along with room temperature Grace's + 5% FBS to total final culture volume of 20mL.

Incubate culture at room temperature until you reach late logarithmic growth. Culture morphology should still resemble heterogeneous small white clumps and lack obvious mycelial threads/tangles.

Transfer at least 20mL of in vitro culture to sterile and spin 3500 rpm, 0h 15m 0s, 4C. This will produce a somewhat loose pellet.

Decant most of supernatant, leaving loose pellet behind.

Resuspend the cells in any remaining supernatant (gently flick tube or pipette up and down with micropipettor) and transfer cell sludge to

Spin eppendorf tube for0h 5m 0s at maximum rpm at -80Room temperature. Tight pellet will result.

Aspirate supernatant and discard.

Flash freeze tube contents using liquid nitrogen or dry ice + ethanol bath.

If you are not ready to proceed with DNA extraction, cells should be stored at -80°C.

This DNA extraction is based on methods reported in Bulat et al, 1998

Remove samples from cold bath or -80°C and thaw at -80Room temperature.

In a fume hood, homogenize each sample with a then rinse pestle with Buffer A 400µL Buffer A.

Add13µL of proteinase K ( 20 mg/mL) to each sample to reach 600 ug/mL.

This assumes pellet volume was approximately 20µL. If substantially deviated from this, adjust volume of proteinase K as needed to achieve desired concentration of 600 ug/mL.

Gently pipette suspension and mix further by gentle inversion, then briefly spin to collect all liquid in bottom of tube.

Add 4.3 uL RNase A to each sample, mixing thoroughly by pipetting or gentle inversion, then briefly spin to collect all liquid in bottom of tube.

Incubate 0h 30m 0s at37°C.

Store at 4°C 0h 30m 0s.

Retrieve samples from fridge and put in chemical safety hood. Allow to come to 4Room temperature before continuing.

Adjust salt concentration to 1 M NaCl of each sample by adding 4.4M NaCl 104µL 4.4M NaCl, mixing gently by inversion.

Add of 24:1 chloroform:octanol 500µL of 24:1 chloroform:octanol to each sample and invert to mix.

Incubate 0h 15m 0s at 4Room temperature.

Spin 0h 2m 0s, 12,000xg, 4Room temperature

Remove aqueous layer (i.e. top layer) and transfer to new tube.

If volume in new tube less than 500µL, add 1 M NaCl to bring up to 500µL.

Sticky aqueous layer is a good sign!

Repeat steps 18) to 21) as necessary until you arrive with a non-cloudy aqueous layer. This may need to be repeated several times.

Spin out any residual protein via spin for 0h 2m 0s, 12,000xg, 4Room temperature.

Using a WIDE BORE TIP (cut P200 tip at first line with clean razor blade to generate wide opening) , transfer supernatant to new tube.

Determine volume of supernatant using gradations on side of tube in combination with pipetting with wide-bore tip, as needed.

Add of glycogen 3µL of glycogen () to solution and mix gently by rocking back and forth.

Add0.6vol of isopropanol (IPA) to supernatant. Invert GENTLY to mix and incubate at RT for 0h 30m 0s.

Spin 0h 10m 0s ,max rpm, 4Room temperature to pellet out DNA.

Wash pellet with of 70% ethanol. 1mLof 70% ethanol.

Spin 0h 2m 0s, max rpm, RT to re-pellet.

Remove all supernatant with P1000/P200 and air-dry ~0h 10m 0s on kimwipe.

WITH WIDE BORE TIP (cut P200 tip at first line with clean razor blade to generate wide opening) resuspend each pellet in desired volume TE buffer (50µL-100µL).

Check polysaccharide and RNA contamination

Equipment

Value	Label
NanoDrop™ One UV-Vis Spectrophotometer	NAME
spectrophotometer	TYPE
Thermo Scientific	BRAND
ND-ONE-W	SKU
Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm	SPECIFICATIONS

1. Apply 1.5µL-2µL of gDNA preparation to nanodrop and read A260/280 (RNA contamination); A260/230 (polysaccharide contamination).

We want to see:

• OD260/OD280 ratio between 1.8 and 2.0 (low protein contamination).
• OD260/OD230 ratio between 2.0 and 2.2 (low carbohydrate contamination). Nanodrop will also give you ballpark for concentration to help select appropriate dilutions for Qubit in next step.

Check DNA concentration using

Equipment

Value	Label
Qubit 2.0 Fluorometer instrument	BRAND
Q33226	SKU
with Qubit RNA HS Assays	SPECIFICATIONS

Prepare triplicate 1/10 and 1/100 dilution samples for Qubit per manufacturer protocol.

Check size distribution

This can be done via agarose gel or

Equipment

Value	Label
4200 TapeStation System	NAME
Electrophoresis tool for DNA and RNA sample quality control.	TYPE
TapeStation Instruments	BRAND
G2991AA	SKU

Gel method:

1. Prepare 0.4% agarose gel in 1x TAE with NO intercalating agent.
2. Use the highest grade agarose you have access to!
3. Add back water after microwaving to maintain correct percentage of agarose.
4. Run out sample with high molecular weight fragment ladder (e.g.) in 1x TAE at 3V/cm at RT until dye reaches ¾ way through gel (expect to take several hours (~4-5). Keep DNA in fridge/on ice during this time (DO NOT FREEZE).
5. Stain with in water (1x solution) for 15 minutes at RT with gentle shaking.
6. Destain with water for 15 minutes at RT with gentle shaking.
7. Image with gel doc.

Tape Station method:

1. Bring & from 4C to RT for at least 30 min before starting.
2. Prepare 10-100 ng/uL dilution of your DNA.
3. Follow TapeStation Genomic DNA ScreenTape instructions to load samples and run.

You could also use the

Equipment

Value	Label
2100 Bioanalyzer Instrument	NAME
Sizing, quantification, and sample quality control of DNA, RNA, and proteins on a single platform	TYPE
Agilent Technologies	BRAND
G2939BA	SKU
http://www.agilent.com/home	LINK

for this analysis, but it's a pain in the butt compared to the TapeStation.