TITLE: Dry Tissue sampling and DNA extraction with NEB monarch Kit
AUTHORS: Alexander  Bradshaw

[STEPS]
?. [Tissue Sampling and Physical tissue disruption]
Flame sterilize a pair of fine-tip forceps in 70% ethanol
?. [Tissue Sampling and Physical tissue disruption]
With the sterilized forceps remove and weigh 10-15mg of Hymenium tissue into a bead bashing tube with a conical bottom (our bead beater cannot hold flat bottomed tubes)*Record the exact weighed amount of sampled tissue in the DNA extraction google sheet
?. [Tissue Sampling and Physical tissue disruption]
fill out a tissue sampled sheet for the specimen and place it back into the collection box. If possible place the tissue sampled from along with the form into a small plastic bag separate from the rest of the specimen.
?. [Tissue Sampling and Physical tissue disruption]
Place 1 [3mm] ball bearing and 8 [1.5mm] into the bead bashing tube with the sampled tissue
?. [Tissue Sampling and Physical tissue disruption]
Place tubes into Bead bug and shake for 30 seconds at 350 speed
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?. [Tissue Sampling and Physical tissue disruption]
Repeat step 5. *if the sample is not a fine powder after a second shake, you may repeat shaking a third time.
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
add 400ul of Monarch tissue lysis buffer to the tube with powdered sample
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
add 10ul of Monarch ProK to sample tube
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
Spin down briefly so liquid and tissue are not stuck on the side of the tube. Place on ThermoMixer at 56C @1350RPM for 30 minutes.
33
56 °C
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
Centrifuge at max speed for 5 minutes
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
*Optional*If tissue or taxa are particularly recalcitrant, a final concentration of   3ug/ml Trichoderma enzymes can be added to the tissue and lysis buffer and incubate at room temperature on the ThermoMixer BEFORE ProK  for 30 minutes
25 °C
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
Pipette supernatant into a new low-retention 1.7ml centrifuge tube.
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
add 4.5ul of NEB RNAse enzyme to sample tube. Incubate on ThermoMixer at 56C and 1350RPM for 15 minutes.Also, place ~1ml of NEB elution buffer (EB) into a centrifuge tube and incubate at 56C, this will be used at a later step.
56 °C
33
?. [Chemical lysis and incubation with NEB monarch Genomic DNA kit reagents]
Centrifuge at max speed for 5 minutes.
?. [GDNA spin column and Elution]
Place 300ul of supernatant into anew tube. Add 600ul (2x volume of supernatant) of NEB GDNA binding buffer.
?. [GDNA spin column and Elution]
Pulse vortex each sample tube for ~10 seconds or until the sample is sufficiently homogenized.
?. [GDNA spin column and Elution]
Add 600ul of Sample/Binding buffer solution to GDNA binding column from NEB monarch kit
?. [GDNA spin column and Elution]
Spin for 3 minutes at 1000g (xg and rcf are the same).This step binds the GDNA to the column
Centrifuge: 1000 34
?. [GDNA spin column and Elution]
Immediately spin at max speed for 1 minute.This step clears all the buffer from the column
?. [GDNA spin column and Elution]
Empty or replace the collection tube, then repeat steps 16 through 18 for the remainder of the sample/binding buffer solution from step 15
?. [GDNA spin column and Elution]
Replace the binding column into a new collection tube. Pipette 500ul GDNA wash buffer onto column and centrifuge at maximum speed for 1 minute.
?. [GDNA spin column and Elution]
Dumb excess buffer from the collection tube and repeat step 20, increase spin time to 1 minute and 30 seconds.
?. [GDNA spin column and Elution]
Place the column into a low-retention 1.7ml centrifuge tube. Add 100ul of 56C elution buffer (EB) from step 12 onto the column. Let incubate on the bench for 1-2 minutes.
25 °C
?. [GDNA spin column and Elution]
Centrifuge sample at maximum speed for 1 minute to elute DNA.
?. [DNA quality control, recording and storage]
Take samples to Nanodrop. Check that the sensor is clean if it is not, place 5ul DNAse free water onto the sensor, and wipe clean with a kimwipe.
?. [DNA quality control, recording and storage]
Blank the nanodrop with 2ul of EB from step 12.
?. [DNA quality control, recording and storage]
Wipe liquid from sensor and pipette 2ul sample onto the sensor and hit "Measure".*make sure you wipe the liquid off of the sensor with a kimwipe every time you measure a new sample or blank and again once finished.
?. [DNA quality control, recording and storage]
Record  these measurements into the google doc for each sample:[ng/ul]: Tells us the concentration of our genomic DNA extractTotal ng: This is the total mass of our extract  (calculate as ng/ul * 100)260/280: Tells us the purity of our sample (DNA ~ 1.8, RNA ~2). a value of ~1.8-2.2 is considered usable260/230: Tells us the cleanliness of our sample. Clean samples are generally ~2.0-2.2.  This value may not be critical if you are using this sample for PCR.Notes: anything you noticed about the spectra curve (also note if it looks normal) from the nanodrop or anything you notice about the sample itself.
?. [DNA quality control, recording and storage]
Place a tough tag on top of the sample tube. Record the catalog number (include institution code if it is not already part of the catalog number)  on the tough tag as well as on the side of the tube.
?. [DNA quality control, recording and storage]
Place the recorded sample into a sample box and store it @ -4C (The cold room).