Integra Magbead DNA and RNA Extraction for isolated colonies

Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Cristina Tato, Jessica Manning, Vida Ahyong

Published: 2022-09-07 DOI: 10.17504/protocols.io.dm6gpjeqjgzp/v1

Abstract

This protocol is the process to extract DNA and RNA from isolated colonies. The extracted high-quality DNA or RNA are suitable for Next-Generation Sequencing (NGS).

Steps

Buffer Preparation

Add 20mL isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
Add30mL isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
Reconstitute lyophilized Proteinase K at 20mg/mL with Proteinase K Storage Buffer and mix by vortexing. Use immediately or store at -20°C .
Reconstitute each vial of lyophilized DNase I with 2.25mL DNase/RNase-Free water in a conical tube.

Note

For each sample to be treated, prepare DNase I Reaction Mix (scale up proportionally):Add 45µL DNase I (reconstituted) and 5µL DNA Digestion Buffer in a nuclease-free tube.mix by gentle inversion and place On ice until ready to use.

Make buffer plates prior to starting protocol

Pre-make Lysis Buffer plate with 520µL DNA/RNA Lysis buffer in 1ml deep well plate.
Pre-make Beads plate with 35µL ZymoBIOMIC MagBinding Beads into 96 V-well PCR plate.

Note

For the Beads plate, make it immediately before starting, <1h prior to starting the protocol, to ensure the beads are kept in suspension.

Pre-make DNA/RNA Wash 1 plate with 520µL MagBead DNA/RNA Wash 1 into 1ml deep well plate. Make it two plates.
Pre-make DNA/RNA Wash 2 plate with 520µL MagBead DNA/RNA Wash 2 into 1ml deep well plate. Make it two plates.
Pre-make 100% Ethanol plate with 1100µL of 100% Ethanol into a 2ml deep well plate. Make it three plates.
Pre-make Prep Buffer plate with 520µL DNA/RNA Prep Buffer into a 1ml deep well plate.
Pre-make water plate with 60µL Nuclease-free water in a 96 V-well PCR plate. Make it two plates.
Spin all plates down for 0h 1m 0s except for the bead plate. Perform a quick pulse spin down of the bead plate, just enough to get all the liquid down. Centrifuge the rest of the plate at 12 000 rpm for 0h 1m 0s .

Sample preparation and Proteinase K

Create a plate map so you know which sample you are adding to each well. Add 50µL of isolated colonies samples to plate 1 (leave column 12 for water control).
Top up the 1x DNA/RNA Shield to get 750µL .
Manually add 120µL of Proteinase K into the 0.2ml 8-strip well.
Use multichannel pipet to add 10µL of Proteinase K into each sample and mix (plate 1).
Load a set of Integra tips (tip set 1) onto the Integra.
Program: Pipet/Mix 250ul, 15 cycles, speed 4. Program the Integra to pipet 250µL of your samples up and down for 0h 1m 0s (15 cycles), then incubate at Room temperaturefor 0h 30m 0s . Keep tips.

Sample binding and washing

Program: Pipet 250ul. Add 500µL total of Lysis Buffer to the sample plate (plate 1).
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix samples and buffer for 0h 2m 0s . Keep tips.
Aliquot 35µL of MagBinding Beads into 96 V-well PCR plate.
Program: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended.
Program: Pipet 30ul. Add 30µL of MagBinding Beads into the sample plate (plate 1).
Program: Pipet/Mix 250ul, 30 cycles, speed 3. Program the Integra to mix the sample and MagBinding Beads plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for 0h 20m 0s .
Transfer the plate/tube to the magnetic stand for 0h 5m 0s until beads (DNA) have pelleted, transfer the cleared supernatant (RNA) into a new 96 V-well plate.

DNA Purification (Beads)

Change new Integra tips.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL MagBead DNA/RNA Wash 1 into sample plate and mix well.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 1 buffer with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL MagBead DNA/RNA Wash 2 into sample plate and mix well.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 2 buffer with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Change new Integra tips.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL 100% Ethanol into sample plate and mix well.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix 100% Ethanol with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Repeat step 24.
Dry the beads for 0h 10m 0s on the magnetic stand.
Change new Integra tips.
Program: Pipet 30ul, speed 5. Dispense a total of 30µL nuclease-free water into the sample plate.
Program: Pipet/Mix 20ul, 30 cycles, speed 7. Program the Integra to mix nuclease-free water with the beads. Keep tips.
Program: Manual Pipet 30ul, speed 3. Transfer the plate to the magnetic stand and pellet the beads for 0h 5m 0s , then aspirate and dispense the eluted DNA to a new 96 V-well plate.
Store DNA sample immediately at -80°C .

RNA Purification (Supernatant)

Change the new Integra tip.
Program: Pipet 230ul, 3 times, speed 7. Dispense a total of 690µL 100% Ethanol to the supernatant.
Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix 100% Ethanol with the supernatant. Keep tips.
Aliquot 35µL of MagBinding Beads into 96 V-well PCR plate.
Program: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended.
Program: Pipet 30ul. Add 30µL of MagBinding beads into the sample plate.
Program: Pipet/Mix 250ul, 10 cycles, speed 3. Program the Integra to mix the sample and MagBinding beads

plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for 0h 10m 0s .

Transfer the plate to the magnetic stand for 0h 5m 0s until beads have pelleted, then discard the cleared supernatant.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL MagBead DNA/RNA Wash 1 into sample plate.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 1 buffer with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL MagBead DNA/RNA Wash 2 into sample plate.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 2 buffer with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL 100% Ethanol into the sample plate.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix 100% Ethanol with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Repeat step 51.
DNase I treatment, use multiple channel pipet to transfer 50µL of DNase I Reaction Mix and mix gently for 0h 10m 0s .
Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500µL DNA/RNA Prep Buffer into sample plate.
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the DNA/RNA Prep Buffer with the beads. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Repeat step 57 to 60.
Program: Pipet 30ul, speed 5. Dispense a total of 30µL nuclease-free water into the sample plate.
Program: Pipet/Mix 20ul, 30 cycles, speed 7. Program the Integra to mix nuclease-free water with the beads. Keep tips.
Program: Manual Pipet 30ul, speed 3. Transfer the plate to the magnetic stand and pellet the beads for 0h 5m 0s , then aspirate and dispense the eluted RNA to a new 96 V-well plate.
Store RNA sample immediately at -80°C .