HMW DNA extraction for amphipods

Prepare the lysis buffer by adding 3 µL of RNase A (100 mg/mL) to 1.5 ml of Buffer G2 per sample.

place live or flash-freezed amphipod in 2ml douncer and add 1 ml of lysis buffer

gently up and down 10 times with the piston

transfer the lysate to a 2 ml tube

rinse the douncer with 0.5 ml lysis buffer and transfer to the 2 ml tube

Incubate for 30 minutes at 37°C.

37°C 0h 30m 0s

Add 75 µL of Proteinase K (20 mg/mL) and incubate with inversion at 50°C for 2 hours.

2h 0m 0s 50°C

Gently transfer in a new 2 ml tube the supernatant with a 1000 µl wide-bore tip , avoiding touching the pelleted debris by gravity.

Place a genomic-tip 20G column on a 15 ml tube

Equilibrate the column with 1 mL Buffer QBT. Wait for the Genomic-tip to drain by gravity.

Carefully apply the lysate to the Genomic-tip with a 1000 µl wide-bore tip. Wait for the Genomic-tip to drain by gravity.

Wash the QIAGEN Genomic-tip with 3 x 1 mL Buffer QC.

Elute the DNA into a new 15 mL tube with 2 x 1 mL of Buffer QF prewarm to 50°C

Add 1.4 mL of room-temperature isopropanol to the éluate, mix by inversion about 10 times, and centrifuge for 30 min at 5,500g at 4°C to pellet DNA. set centrifuge deceleration speed to level3

5500x g,4°C

Gently remove the supernatant using a P1000

Gently resuspend the pellet with 1 ml of cold 70% ethanol using a 1000µl wide-bore tip.

Transfer to a new 1.5 ml tube.

Centrifuge for 10 min at 5,000g at 4°C to pellet DNA. set centrifuge deceleration speed to level3

5000x g,4°C

Remove as much supernatant as possible, avoiding touching the pellet.

Air dry the pellet 10 min at RT.

0h 10m 0s``Room temperature

Resuspend the pellet with 50 µL of TE 1x prewarm to 50°C

Incubate for 1hour at 50°C, then overnight at RT.

1h 0m 0s 50°C

Room temperature

Quantify your sample with a Qubit HS.

Analyse 1 µL in a UV spectrophotometer (e.g. Nanodrop).

Visualise 1 µL of sample to estimate the molecular weight. (Tapestation)

QC results of extraction of 4 individuals

A	B	C	D	E
63	39.4	1.82	1.86	57
40	30.9	1.88	2.20	57
65	82.1	1.78	1.82	50
43	197	1.86	2.33	26

Tapestation profiles