TITLE: Genomic DNA extraction from animal faecal tissues using DNeasy blood and tissue kit
AUTHORS: Somasundhari Shanmuganandam, Benjamin Schwessinger, Robyn Hall

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Faecal tissues are difficult to lyse and contain lower amount of DNA compared to other animal tissues such as liver and duodenum. This protocol was optimised to extract maximum amount of DNA using 50 mg of faecal tissues following Qiagen DNeasy blood and tissue kit.</div></div>

[STEPS]
?. [Tissue subculturing]
Thaw the samples in ice and bring it to room temperature
?. Using a scapel, place the tissue in a petri dish and cut 50 mg of tissue. Check the tissue weight to confirm. Place it in a 2 ml eppendorf tube filled with 0.5 cm of purified silica beads.
?. [DNA extraction]
Add 324 μl of buffer ATL and 36 μl of proteinase K into each of the bead tubes containing sample.For reagent only control, take empty bead tube and add only buffer ATL and proteinase K.
?. [DNA extraction]
Place the bead tubes into the bead beater for 2 x 20 sec and spin it down.
?. [DNA extraction]
Incubate the samples at 56°C in heat block for 10 min to 30 min. Actual time depends on tissue lysis
?. [DNA extraction]
Add 4 μl of RNase A (100 mg/ml) to the lysed samples, vortex and incubate for 5 min at room temperature.
?. [DNA extraction]
Vortex and transfer 200 μl of the sample solution into a new 1.5 ml eppendorf tube.
?. [DNA extraction]
Transfer the supernatant (as much as possible) into a new 1.5 ml eppendorf tube and discard the pellet.Note: The supernatant may contain tiny tissue fragments which can be lysed in the next step
?. [DNA extraction]
Add 200 μl of buffer AL and vortex.
?. [DNA extraction]
Add 200 μl of ethanol pipetting up and down. Vortex.Note: Step 10 must be done immediately after step 9
?. [DNA extraction]
Tranfer the entire content of the eppendorf tube into a spin column provided with the kit.
?. [DNA extraction]
Centrifuge at 6,000 rcf for 1 min. Discard the flow through and collection tube.
?. [DNA extraction]
Place a new collection tube under the spin column and add 500 μl of buffer AW1
?. [DNA extraction]
Centrifuge at 6,000 rcf for 1 min. Discard the flow through and collection tube.
?. [DNA extraction]
Place a new collection tube under the spin column and add 500 μl of buffer AW2.
?. [DNA extraction]
Centrifuge at maximum speed (13,000 rcf) for 3 mins
?. [DNA extraction]
Discard the flow through and place the same collection tube back into the column. Perform centrifuge for 1 min at maximum speed
?. [DNA extraction]
Discard collection tube and place a 1.5 ml eppendorf tube under the column and add 100 μl of buffer AE. Let it stand for 1 min.
?. [DNA extraction]
Centrifuge at 6000 rcf for 1 min.
?. [DNA extraction]
Pipette the eluted DNA from the eppenforf tube and add it into the column again for re-elution
?. [DNA extraction]
Let it stand for one min and centrifuge at 6000 rcf for 1 min.
?. [DNA extraction]
Discard the filter column. Store 50 μl of eluted DNA at 4°C for quantitative analysis and store the rest at -20°C to prevent repeated freeze-thaw cycle.
?. [Gel electrophoresis]
Prepare 0.8% agarose gel and run at 100 V for 40-45 min to check the quality of DNA extracted
?. [Qubit analysis]
Make sure the qubit is standardised prior to starting
?. [Qubit analysis]
Use qubit broad range DNA assay kit and perform analysis using 1 μl of extracted DNA. Note down the concentration value (ng/μl)
?. [Nanodrop]
Use 2 μl of distilled water as blank and 2 μl of buffer AE as standard. Clean the surface between each read.
?. [Nanodrop]
Add 2 μl of sample DNA into the surface of the nanodrop and read. Clean the surface before next use. Note down the A260/280, A260/230 and concentration (ng/μl)