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At end of day, remember to enter amount of PVP, B-merc and chloroform used.

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a. Computer p/w: NParks123&2022

b. Go to sequencing logs and open the excel.(command F, “sequencing logs”)

c. Fill in data (get the GK sequencing code and note them down).

d. Open any “sequencing order form”, fill in as needed.

e. Save as a new copy and email to Mui Hwang.

f. Prepare 3 μl of each primer for each sample (i.e. 5 samples = 15 μl of each primer)

g. take out Sample collectino card and write in the details.

h. place in the eppendord tubes and the primers into the collection card. When done, place the collection card into ziplock bag.

i.  place it in freezer to send for sequencing.

j. At end of day, make sure to record down amount of afarose powder, florosafe and number of collection tubes used.

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(For samples with no DNA strain, no need to excise the ge;. for those with double strain, take out both strains.)

a. Prepare a 55°C water bath.

b. Excise the agarose gel with a clean plastic cutter

c. transfer the gel into labelled eppendorf tube

d. Add 500 μl of FADF buffer to the sample and mix by vortex

e. Incubate at 55°C water bath, vortex every 2-3 mins till the gel dissolved.

f. Cool the sample to room temperture.  while waiting place a FADF column into a collection tube and lebel tubes.

g. Transfer 800 μl of the sample mixture into the FADF Column and centrifuge at 11,000 x g for 30 seconds. Discard flow into empty beaker.

h. Add 750 μl of the wash buffer (ethanol). Centrifuge at 11,000 x g for 30 seconds. Discard flow into beaker.

i. Add 750 μl of the wash buffer (80% ethanol). Centrifuge at 11,000 x g for 30 seconds. Discard flow into beaker.

j. Centrifuge tubes again after discarding flow, centrifuge the tubes for 3 minutes to dry.

k. Transfer FADF solumn into labelled eppendorf tube (label using the GK labels).

l. Add 40ul of elution buffer  and let it stand for 1 minute.

m. Centrifuge eppendorf tube at 17,900xg for 1 minute.

n. throw away FADF column and cap the eppendorf tube properly.

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a. Place gel onto UV tray and turn on the computer.

b. Launch “Image Lab 4.0”, select “new”. Under nucleic acid gels → “gelred”. Deselect “highly saturated pixels”.

c. “Position gel”

d. “Run Protocol”

e. Printing: “Image tools” → Crop, Paper size 1280 x 1920

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i. Weigh agarose powder into a non-autoclaved conical flask (medium tray: 3.2g, small tray, 2g)

ii. Measure the required volume of 1x TAE into the conical flask + 20 ml (excess) + 10ml (account for evaporation in the microwave) (medium tray: 170ml, small tray, 110ml)

iii. Place the conical flask into microwave for around 5 mins till all powder is dissolved.

iv. Remove the conical flask from the microwave and wrap it in a C-fold towel that has been wet with ethanol till warm to touch. Swirl the conical flask to mix and to ensure solution does not solidify.

v. Add Florosafe DNA stain (4 μl / 100 ml of buffer) (Medium tray: 6.4ul, small tray: 4.4ul)

vi. Wait around 45 mins for gel to set

vii. When gel solidified, transfer the tray to the electrophoresis machine, ensuring wells are at the black end. Top up with 1x TAE buffer if needed.

viii. Load 3 μl of DNA ladder and load wells with samples (25 μl).

ix. Make sure that machine is set to 70 volts, 700 current and 75 minutes. when done, run and machine and make sure bubbles are visible from the sides.

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a. Refer to the checklist for size of gel trays – use comb size 32.34 μl as PCR product is 25 μl, and requires an agarose gel of at least 10 cm runway.

b. Disinfect gel tray, mould and comb with ethanol. Ensure the tray is levelled.

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Steps for last day extraction of samples

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Steps for last day extraction of samples

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c. Pipette 24 μl of master mix and 1 μl of DNA to labelled PCR tubes = Total 25 μl
d. Shake and spin down.
e. place tubes into PCR machine and let it run.
temperature , time for each step and cycles are as shown above.

When PCR is done in time, place tubes inside freezer. If not the tubes can stay inside PCR machine overnight.

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See number of samples and calculate the respective amount of water, Accustart and primers needed for master mix. Mastermix volumes are as shown above.

After done, shake and spin down the mastermix.

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i. Collect primers from freezer using an icebox (ITS4, ITS1F, LROR, LR7).
ii. unfreeze them
iii. Pipette 10 μl of primer and 90 μl of autoclaved water into a tube (10x dilution)

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4. PCR (Acc of AccuStart II GelTrack PCR SuperMix protocol

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4. PCR (Acc of AccuStart II GelTrack PCR SuperMix protocol

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i. Launch the Nanodrop programme, select “Nucleic Acid”
ii. Clean the reader with kimwipes and milli-Q water
iii. Add TE buffer and press the "blank button".
iv. Rename sample inside the system and measure the nucleic acid concentration fior all samples.
v. After done, save it under Serena folder and ake a picture as record.

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i. Remove the Eppendorf tubes with DNA out of the water bath and do a quick spin. Transfer the DNA to labelled PCR tubes.

ii.Vortex the SPRI magnetic beads which the DNA will stick onto

iii. Pipette 25 μl of beads to each PCR tube. Cap, shake, wait for 5 mins and quick spin.

iv. Place the PCR tubes in the magnetic rack and wait till the solution turns clear.

v. Pipette out the supernatant by pointing the pipette tip to the opposite side of the magnetic beads and sucking everything out.

vi. Add 100 μl of 80% ethanol to each PCR tube while hovering the pipette over.

vii. Shake and spin the PCR tubes on a tabletop centrifuge.

viii. Extract the liquid with a micropipette set to 110-120 μl (larger vol than liquid avail so that can get everything)

ix. Repeat vi-viii with 80% ethanol (once), followed by 99% ethanol (once). Use the 10 μl pipette to extract every last drop.

x. Pipette 25 μl of TE buffer into the PCR tube by adding onto the beads. Shake the tubes, give a quick spin too. (TE buffer resuspends the DNA).

xi. Wait for 5 mins, table spin again.

xii. Place the PCR tubes on the magnetic rack.

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i. Centrifuge the tubes at max speed for 3 mins. Ensure the lid is closed and tighten. (FYI: if the DNA sample is sticky like mucus, it is a sign of high carb concentration, requiring sorbitol wash)

ii. Pour the liquid into a waste beaker and be careful not to lose the DNA pellet

iii. Pipette 700 μl of 80% Ethanol into each tube and flick or slightly vortex it to mix (This washes out the salts and OM, alcohol conc. Must be above 60% otherwise the DNA will dissolve).

iv. Centrifuge for 1 min at max speed

v. Pour out the liquid into the waste beaker and be careful not to lose the DNA pellet.

vi. Repeat step iii-v above with 80% alcohol (twice), and lastly 99% alcohol (once).

vii. Place the tubes in the speed vac for 20 mins.

viii. Place the tubes in the speed vac for 20 mins
Pipette 25 μl of autoclaved water into the tubes by inserting the pipettes into the Eppendorf tubes and carefully aiming it at the DNA pellet. Place the tubes in a 55°C water bath for 1 hour to resuspend the DNA.

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Steps for second day eatraction of samples

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Steps for second day eatraction of samples

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i. use 1000ul micropipette and find the tube with the largest volume of DNA and measure the estimated volume of DNA by adjusting the pipetting accordingly.

ii. Once the volume is obtained, pipette 0.08x the volume of Ammonium acetate (in fridge) into each tube.

iii. Of the combined volume (original volume + ammonium acetate), add 0.54x the volume of isopropanol (ammonium acetate and isopropanol helps to precipitate the DNA).

iv. Mix well but gently with a micropipette and quick spin to remove air bubbles

v. Place the tubes into the freezer overnight.

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i. Remove the tubes from the water bath and spin down the solution

ii.  Wear nitrile gloves and obtain chloroform from the corrosive cabinet and bring it to the fumehood.

iii. Transfer 600 μl of chloroform using 1000ul micropipetteto each tube. Pour some into a beaker for easier pipetting. Avoid dripping the chloroform on any other surfaces as it is corrosive and non-adhesive.

iv. Cap the tubes and shake carefully.

v. Centrifuge for 8 minutes. Make sure the units are rcf/xg and the contents should be separated into 3 layers.

vi. Within the fumehood again, use a 200 μl pipette (for gentler suction), take out the top aqueous DNA layer as much as possible. Be careful to not get the chloroform, especially when there are leakages in the layers.

vii. Empty the chloroform in old tubes into the waste container and dispose the old tubes and pipette tips into the waste bin

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After 2 days, take out specimens and use disposable pestle and smash the mushroom specimen till powdery consistency is reached.

After that, top up with additional 20ul of CTAB.

Place the tubes into water bath for 2 hours at 55°C

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if the quantity of specimens is too small, soaking of samples in CTAB will be done.

Add in 500ul of CTAB into clean labelled disposal mortar and pestle tube and let it rest for 2 days.

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Specimens that require use of mortar and pestle.

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Specimens that require use of mortar and pestle.

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1b. collecting and preparing of mushroom specimen.

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1b. collecting and preparing of mushroom specimen.

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i. take out thermoflask and add in a suitable amount of liquid nitrogen (make sure to wear gloves when handling liquid nitrogen).

ii. Add in mushroom specimen inside eppendorf tube into autoclaved mortar and pestle. Using the autoclaved mortar and pestle, add 2 scoops of liquid N. This cools it down and preps it for the specimen. Liquid N keeps the specimen cool so that it does not become mushy and becomes easier to grind.

iii. Grind the specimen till powdery.

iv. using disposable spatula, scrap off the mushroom specimen from the mortar and pestle's surface. After that, add in another scoop of liquid introgen and grind it again.

v. Use the spatula and scrap off all mushroom powder from the surfaces of mortar and pestle. pack the powder into one spot.

vi. Add 600 μl of CTAB buffer using 1000ul micropipete to the mortar. And gently  mix till a liquid consistency is obtained. Place the mortar into a warm water bath to melt the Ctab solution in case it freezes up.

vii. Using 1000ul micropipette, set it to 800ul and transfer the liquid into a clean labelled Eppendorf tube.

viii. Incubate the tubes for 2 hours at 55°C (if water bath is occupied, use the heating block.

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ai. Preheat the water bath to 55°C (must for incubation) or 60°C.

aii. Disinfect the lab bench before working on it

aiii. Take note of the required amount as per the stock recipe as shown above.

aiv. PVP measurement (powder): Fold a piece of weighing paper into a triangle, place it into the weighing machine and tare. Disinfect a metal spatula with ethanol before scooping and weighing the required amount of PVP. Transfer the PVP into a clean falcon tube. Wash spatula after use.

av. CTAB measurement (liquid): Pipette the required amount of CTAB stock into the falcon tube. Ensure that the sides of CTAB borosilicate glass jar is not touched. Transfer the CTAB stock into the falcon tube with care (since pvp is light and can fly out easily).

avi. when both pvp and CTAB is placed inside falcon tube, place it into water bath for around 30 minutes (to change the temperature of the water bath, press and hold the button and turn the knob till teperature is set.

avii. β-merc measurement: [Collect keys from Mui Hwang’s table] Using a micropipette, add the required volume of β-merc into the falcon tube within the fume hood) Swirl the tube to mix.

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Steps for first day extraction of samples

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Steps for first day extraction of samples