From

DNAse I working solution: 70µL RDD + 10µL DNase I working solution per sample

Included within

Proteinase K working solution for DNA isolation: 60µL AW1 + 20µL Proteinase K per sample

Included within

Add 10µL ß-Mercaptoethanol (not included in kit) per 1mL of RLT plus buffer

This protocol is for Sample

Optional: Weigh tissue before start to decide for volume

Enter a complete list of samples used for each experiment below:

A	B	C	D	E

List of tissue sample used in experiment

Keep tubes with tissue on dry ice and make sure that they do not thaw during processing.

Transfer the tissue piece into a 6-well plate or small petry dish placed on dry ice and cut an appropriate piece of tissue (ca. 10-30 mg) with a safety scalpel.

Transfer tissue in 350µL 600µL RLT + ß-ME in 2 ml DNA LoBind tube.

Add a 5mm bead to each tube and lyse tissue in TissueLyser for 0h 2m 0s @ 20 Hz

Equipment

Value	Label
TissueLyser II	NAME
Bead Mill	TYPE
QIAGEN	BRAND
85300	SKU

Spin down for 0h 1m 0s @ 9000x g,0h 0m 0s

Turn tube rack and lyse tissue for 0h 2m 0s @ 20 Hz

Spin down for 0h 1m 0s @ 9000x g,0h 0m 0s

Add lysed product to DNA Mini Spin Column

Spin 0h 0m 30s @ maxx g,0h 0m 0s , repeat if any liquid remains on column

Transfer column to a new collection tube and store tube at 4°Cuntil DNA extraction

Transfer flow-through to new 2 ml LoBind tube

Add 50µL 80µL Proteinase K (not diluted), mix by pipetting

Add 200µL 350µL EtOH abs., mix by inverting, spin down liquid

Incubate 0h 10m 0s @ Room temperature

Add 400µL 750µL EtOH abs. , mix by pipetting

Add 700µL to RNeasy spin column and spin for 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

Repeat until all liquid passed through the column

Add 500µL RPE

Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

Add 80µL DNase I working solution directly onto the membrane and incubate 0h 15m 0s at Room temperature

Add500µL FRN

Spin 0h 0m 30s @ maxx g,0h 0m 0s and don't discard flow-through and place column in new collection tube

Add flow-through again to column

Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

Add 500µL RPE

Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

Add 500µLEtOH abs

Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new collection tube

Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new 1.5 ml collection tube

Add 30µLof RNase-free H₂O

Incubate 0h 1m 0s @ Room temperature

Spin down0h 1m 0s @ 8000x g,0h 0m 0s

QC: Qubit and tape station

350µL AW1 auf DNA Mini Spin Column geben

Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

80µLProteinase K working solution directly onto membrane

Incubate 0h 5m 0s @ Room temperature

Add 350µL AW1

Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through

Add 350µL AW2

Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new 2 ml collection tube

Spin 0h 1m 0s @ maxx g,0h 0m 0s and place column in new 1.5 ml collection tube

Add 50µL directly onto membrane

Incubate 0h 1m 0s @ Room temperature

Spin down 0h 1m 0s @ 8000x g,0h 0m 0s

QC: Qubit DNA and tape station